CN118076629A - KCNV2 variants and uses thereof - Google Patents

Abstract

Disclosed herein are novel polynucleotide variants of KCVN2, voltage-gated potassium channel polypeptides encoded thereby, and their use, e.g., in methods of restoring photoreceptor function and treating subjects suffering from retinal disorders such as CDSRR.

Description

KCNV2 variants and uses thereof

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 63/191,106, filed 5/20 at 2021, which is incorporated by reference in its entirety.

Technical Field

The present application relates to novel nucleotide and protein sequences (such as KCNV sequences), as well as recombinant nucleic acid molecules and vectors, and related methods for treating retinal disorders (such as cone cell dystrophy) in a subject.

Background

Cone cytodystrophy, such as but not limited to cone cytodystrophy with abnormal rod cell response [ CDSRR ], is an autosomal recessive disorder that may be characterized by, for example, poor visual acuity, vision loss, sensitivity to light, poor color vision, nystagmus, strabismus, and the like. Vision difficulties begin in early childhood and vision is, for example, 20/100 or less by the age of twenty years. Patients may later develop night blindness and many or most patients may also develop myopia. There is no specific treatment to reduce or prevent progression of vision loss, which results in poor prognosis for patients and reduced quality of life as they age. Thus, there is a need to determine treatment methods.

Disclosure of Invention

We disclose novel recombinant nucleic acid molecules, proteins, vectors and related methods for treating cone dystrophy [ CDSRR ] (retinal cone dystrophy 3b, omim 610356) and related pathologies indicated by KCVN2 with aberrant rod cell responses. CDSRR is a rare recessive inherited retinopathy characterized by poor visual acuity (due to central dark spots), photophobia, severe color vision defects, and occasionally also nystagmus and strabismus. In some patients, the fundus appears normal, but foveal or parachlorogenic atrophy, macular bulls eye, hyper-fluorescent abnormalities, and extensive fine pigmented retinopathy have been reported. There may be some temporal pallor in the optic nerve.

Disclosed herein are nucleic acids, transcription Control Units (TCUs), optimized gene sequences, expression constructs, and vectors for expressing genes in retinal cells, including but not limited to cone cell photoreceptors and/or rod cell photoreceptors.

Disclosed herein are modified KCVN genes containing a nucleic acid substitution in the unmodified KCVN2 gene, wherein the nucleic acid substitution may be one or more of those displayed by aligning the human KCVN gene with the codon optimized version represented by SEQ ID NO 2.

Nucleic acids, transcription Control Units (TCUs), optimized gene sequences, expression constructs, and vectors for expressing genes in photoreceptors (e.g., cone cell photoreceptors and/or rod cell photoreceptors) are also provided.

Also provided are vectors, such as adeno-associated virus (AAV) vectors, comprising the nucleic acid molecules, and isolated kv8.2 proteins encoded by the nucleic acid molecules.

Also provided are expression constructs comprising the variant human KCVN gene under the control of TCU. In one variation KCVN is under the control of a promoter optimized for expression of the gene in a photoreceptor (e.g., a cone cell photoreceptor or a rod cell photoreceptor). In one variant, the variant human KCVN gene may be under the control of a Rhodopsin Kinase (RK) promoter.

Thus, in one variation, we provide:

A promoter capable of directing the expression of KCVN genes. In one variant, the promoter targets transgene expression to the photoreceptor. In another variant, the promoter is limited to expression by the photoreceptor only. In another variant, the promoter is a Rhodopsin Kinase (RK) promoter.

Sequences to be expressed in photoreceptors. In one variation, the invention provides an expression construct comprising a sequence to be expressed in a photoreceptor-specific manner. In another variant, the sequence to be expressed comprises a gene encoding kv 8.2. In another variant, the sequence to be expressed comprises SEQ ID NO.2.

The foregoing and other features and advantages of the disclosure will become more apparent from the following detailed description of various embodiments, which proceeds with reference to the accompanying drawings.

Drawings

FIG. 1 shows a schematic representation of a first construct.

Fig. 2 shows a schematic representation of a second construct.

Fig. 3 shows a schematic representation of a third construct.

Fig. 4 shows a schematic of a fourth construct.

Fig. 5 illustrates an exemplary method of restoring retinal function.

Fig. 6 provides a phase I and phase II investigation procedure.

Fig. 7 provides a phase III proof of concept study.

Figure 8 provides a comparison between treated and untreated mice using the disclosed methods, systems and nucleic acid sequences.

Fig. 9 shows the relative gene expression of cone-free (a cone cytostatic protein) and rod cell (B, rhodopsin) markers in Wild Type (WT), uninjected and treated retinas P <0.05; * P <0.01.

FIG. 10 shows representative images of retinal expression of human Kv8.2 subunit in the retina of Kv8.2 KO mice injected with SEQ ID NO:2 delivered by SEQ ID NO:3 12 weeks after treatment.

Fig. 11 is a table showing a protocol for a mouse pilot study.

Figure 12 shows data representing a wave amplitude for treated and untreated mice.

Fig. 13 shows data representing positive b-wave amplitudes of treated and untreated mice.

Fig. 14 shows OCT data for treated and untreated mice.

FIG. 15 shows quantification of c-waves from Wild Type (WT), kv8.2 KO untreated (uninjected) and Kv8.2 KO eyes.

Fig. 16 and 17 show improved photopic and scotopic visual acuity and scotopic contrast sensitivity in kv8.2ko mice treated 12 weeks after treatment.

Fig. 18 is an overview (overview) of retinal sections from subretinal injected eyes with treated regions showing human kv8.2 subunit expression (green) and untreated regions without kv8.2 expression.

Fig. 19 provides higher magnification images showing treated and untreated areas of kv8.2 expression (green), kv2.1 expression (red) and nuclei (blue). Scale bar = 50 μm.

Figure 20 provides data from real-time quantitative PCR showing expression of the human KCNV gene in treated eyes (normalized to wild type).

FIG. 21 together with FIG. 2 shows the sequence alignment of human KCVN (SEQ ID NO: 11) and its codon optimized variant SEQ ID NO: 2.

Sequence listing

The nucleic acid and amino acid sequences listed in the appended sequence listing are shown using standard alphabetical abbreviations for nucleotide bases and three letter codes for amino acids, as defined in 37 c.f.r.1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by reference to the strand shown. The sequence listing is submitted as an ASCII text file in the form of a file named "sequence listing. Txt" (about 40 kb) created at 5.18 of 2022, which is incorporated herein by reference.

Detailed Description

We disclose novel recombinant nucleic acid molecules, proteins, vectors and related methods for treating cone dystrophy [ CDSRR ] (retinal cone dystrophy 3b, omim 610356) and related pathologies indicated by KCVN2 with aberrant rod cell responses. CDSRR is a rare recessive inherited retinopathy characterized by poor visual acuity (due to central dark spots), photophobia, severe color vision defects, and occasionally also nystagmus and strabismus. In some patients, the fundus appears normal, but foveal or paracardial atrophy, macular bulls eye, high fluorescence abnormalities, and generalized fine pigmented retinopathy have been reported. There may be some temporal pallor in the optic nerve.

Clinical symptoms may be limited to vision loss without other tissues or organs being affected. Most governmental agencies define legal blindness as corrected visual acuity (central vision) of 20/200 or less in the eye with best vision. This means that legal blind people can see what is at 20 feet and ordinary people can see clearly at 200 feet. In CDSRR, studies have shown that visual acuity can vary from person to person, but averages about 20/160, but eventually may develop to legal blindness levels. There is no specific treatment available to reduce or prevent the progression of vision loss, which results in poor prognosis for patients and reduced quality of life as they age. Since prognosis is associated with the likelihood of healing, treating or curing a condition, a poor prognosis for CDSRR and other KCVN 2-related pathologies means little chance of recovery, as no cure or therapy is currently available. Amblyopia aids and colored lenses are the only sources that a patient can use to ameliorate symptoms of vision loss. Several studies have shown that CDSRR patients have progressive central vision loss, but extensive natural history studies have not been completed to assess the full extent of disease progression.

The rarity of this disease (estimated 1/1,000,000 of the number of globally infected individuals) makes it a definition of rare disease, which has hampered the pharmaceutical industry to address the disease, as it provides little financial incentive for the private sector to produce and sell new drugs for treating or preventing this disease. However, the availability of Electroretinograms (ERGs) and gene detection allows for accurate and early CDSRR diagnosis and supports the development of therapies for this disease. CDSRR's recessive genetic pattern and slow progression make it a good candidate for disposable virus-based gene therapy (gene supplementation) therapy.

One of the causative agents of CDSRR is a mutation in the potassium channel voltage-gated V subfamily member 2 gene (KCVN 2). In some CDSRR patients, there was a mutation in the KCNV gene encoding the kv8.2 voltage-gated potassium (K ⁺) channel subunit. Currently, over 95 different variants of KCNV have been identified worldwide that cause CDSRR, including missense, nonsense, intragenic deletions, extraframe insertions, and the like. It has been shown that different variants have different effects on kv8.2, some of which produce non-conducting channels, while others completely prevent the formation of channels. This suggests that there are different mechanisms involved in disease pathology, but also that all variants produce a non-functional protein, which makes CDSRR a good candidate for gene replacement therapy.

Kv8.2 is a member of a group of "modified/silent" channel proteins that do not form channels themselves, but require a cognate partner; for kv8.2, this is kv2.1 (encoded by KCNB 1), a member of the Shab subunit family, which produces a delayed rectifier current that regulates the repolarization rate of the action potential. In the eye, the kv8.2 and kv2.1 subunits are located only on the cytoplasmic membrane of the inner segment of cone cells and rod cell photoreceptor cells, which are responsible for initiating the light transduction cascade of the visual response. However, the missense mutation of KCNV also has been shown to lead to human epilepsy, suggesting that it may also be expressed in the brain. Electroretinogram (ERG) disease phenotype suggests that mutations in KCNV lead to loss of function of the kv8.2 subunit, resulting in loss of function of the kv2.1/kv8.2 heteromers. Ultimately, this will alter the sensitivity of the retina to light, thereby altering the underlying physiological process of adjusting the dynamic range of vision at different illumination levels.

There is evidence that the KCNV mutations affect both cone and rod photoreceptors, which are reflected in abnormal ERG recordings of both photoreceptors and are widely distributed throughout the retina, however, in some patients, the observed morphological changes appear to be more pronounced in cone cells. High resolution imaging of the retina using spectral domain optical coherence tomography (SD-OCT) in CDSRR patients reveals that severe morphological abnormalities typically occur in the central retina. These include internal/external segment (IS/OS) junctions, significant foveal depth reduction, disruption of cones photoreceptor chimerism with plaques of cones loss, and overall reduction in cones density. However, the mechanism of photoreceptor cell loss in CDSRR retinas remains unknown. Furthermore, it is not clear how the disease affects cone cells differently than rod cells. A recent study of abnormalities in CDSRR patients as measured by pupillometry has shown that retinal internal function can be preserved. In one variation, therapies designed to restore extraretinal function may be successful.

Disclosed herein are nucleic acids, transcription Control Units (TCUs), optimized gene sequences, expression constructs, and vectors for expressing genes in retinal cells, including but not limited to cone cell photoreceptors and/or rod cell photoreceptors.

Disclosed herein are modified KCVN genes containing a nucleic acid substitution in the unmodified KCVN gene (SEQ ID NO: 12), wherein the nucleic acid substitution may be one or more of those displayed by aligning the human KCVN gene with the codon optimized version represented by SEQ ID NO 2.

Nucleic acids, transcription Control Units (TCUs), optimized gene sequences, expression constructs, and vectors for expressing genes in photoreceptors (e.g., cone cell photoreceptors and/or rod cell photoreceptors) are also provided.

Also provided are vectors, such as adeno-associated virus (AAV) vectors, comprising the nucleic acid molecules and isolated kv8.2 proteins encoded by the nucleic acid molecules.

Expression constructs comprising the variant human KCVN gene under the control of TCU (SEQ ID NO: 2) are also provided. In one variation, KCVN2 is under the control of a promoter optimized for expression of the gene in a photoreceptor (e.g., a cone cell photoreceptor or rod cell photoreceptor). In one variant, the variant human KCVN gene may be under the control of the Rhodopsin Kinase (RK) promoter (SEQ ID NO: 6).

Variants of the KCVN gene having increased gene expression relative to the corresponding native human KCVN gene (SEQ ID NO: 12), such as SEQ ID NO:2, are also provided. Variants of KCVN2 genes have improved therapeutic properties, including improved expression, including up to an average about 8-fold increase in gene expression compared to wild-type; higher differences and significant reduction in ERG positive b-wave data compared to untreated eyes; and higher variance and significant reduction in ERG positive b wave data compared to that obtained by other products. Improved properties of the disclosed KCVN2 variants (e.g., SEQ ID NO: 2) include, but are not limited to, increased expression compared to the corresponding native human KCVN gene (SEQ ID NO: 12); increased expression compared to the corresponding wild-type KCVN genome (SEQ ID NO: 12); and/or improved pharmacokinetic properties compared to the corresponding native human KCVN gene (SEQ ID NO: 12). Improved properties may include improved transcript stability and minimal aberrant transcript splicing.

Also disclosed are methods (including but not limited to CDSRR) for treating and/or preventing a retinal disorder or malnutrition using one or more of nucleic acids, transcription Control Units (TCUs), optimized gene sequences, expression constructs, and vectors.

Also disclosed is an AAV-mediated gene amplification therapy for KCNV-related cone dystrophy with an aberrant rod cell response.

Thus, in one variation, we provide:

A promoter capable of directing the expression of KCVN genes. In one variant, the promoter targets transgene expression to the photoreceptor. In another variant, the promoter is limited to expression by the photoreceptor only. In another variant, the promoter is the Rhodopsin Kinase (RK) promoter (SEQ ID NO: 6).

Sequences to be expressed in photoreceptors. In one variant, the invention provides an expression construct comprising the sequence to be expressed in a photoreceptor-specific manner. In another variant, the sequence to be expressed comprises a gene encoding kv 8.2. In another variant, the sequence to be expressed comprises SEQ ID NO.2.

The foregoing and other features and advantages of the disclosure will become more apparent from the following detailed description of various embodiments, which proceeds with reference to the accompanying drawings.

Terminology

Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Krebs et al (eds.), lewis's genes XII, published by Jones & Bartlett Learning, 2017; KENDREW ET al (eds.), the Encyclopedia of Molecular Biology, published by Blackwell Science ltd, 2009 (ISBN 9780632021826). The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. "comprising A or B" is meant to include A, or B, or A and B. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. In case of conflict, the present specification, including definitions of terms, will control. For purposes of facilitating an overview of the various embodiments of the present disclosure, the following explanation of specific terms is provided:

5 'and/or 3': nucleic acid molecules (such as DNA and RNA) are referred to as having a "5 'end" and a "3' end" because a single nucleotide reacts to produce a polynucleotide in the following manner: the 5 'phosphate of one single nucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester bond. Thus, when the 5' phosphate of a linear polynucleotide is not linked to the 3' oxygen of a single nucleotide pentose ring, one end of the linear polynucleotide is referred to as the "5' end". When the 3' oxygen of a polynucleotide is not linked to the 5' phosphate of another single nucleotide pentose ring, the other end of the polynucleotide is referred to as the "3' end". Although the 5 'phosphate of one single nucleotide pentose ring is attached to the 3' oxygen of its neighbors, the internal nucleic acid sequence can also be said to have 5 'and 3' ends.

In linear or circular nucleic acid molecules, discrete internal elements are referred to as "downstream" or "upstream" or 5 'of the 3' element. With respect to DNA, this term reflects that transcription proceeds in the 5 'to 3' direction along the DNA strand. Promoter and enhancer elements that direct transcription of linked genes are typically located 5' or upstream of the coding region. However, the enhancer element can exert its effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

Adeno-associated virus (AAV): small replication-defective non-enveloped viruses that infect humans and some other primates. AAV is not known to cause disease and to elicit a very mild immune response. Gene therapy vectors utilizing AAV are capable of infecting dividing cells and resting cells, and are capable of maintaining an extrachromosomal state without integration into the genome of the host cell. These characteristics make AAV an attractive viral vector for gene therapy. There are currently 11 recognized serotypes of AAV (AAV 1-11).

Administration/administration form: an agent, such as a therapeutic agent (e.g., recombinant AAV), is provided or administered to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, intratubular, sublingual, rectal, transdermal, nasal, vaginal, and inhalation routes.

CDNA (complementary DNA): a DNA fragment lacking internal non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory from messenger RNA extracted from cells by reverse transcription. The cDNA may also comprise an untranslated region (UTR) responsible for translational control in the corresponding RNA molecule.

KCNV2: the human KCNV gene (NCBI reference sequence: NM-133497.4; ensembl gene ENSG00000168263.9 and transcripts KCNV2-201 ENST00000382082.4) (SEQ ID NO: 1) is the transgene of choice. KCNV2 the transcript has only one splice variant and consists of 2 exons. The entire transcript was 2178 base pairs long and the coding sequence (CDS) used as the base for our study was 1638 base pairs long.

Codon optimized: a "codon optimized" nucleic acid refers to a nucleic acid sequence that has been altered such that the codon is optimal for expression in a particular system (such as a particular species or group of species). For example, the nucleic acid sequence may be optimized for expression in mammalian cells or in a particular mammalian species (such as human cells). Codon optimization does not alter the amino acid sequence of the encoded protein.

Control: reference standard. In some embodiments, the control is a negative control sample obtained from a healthy patient. In other embodiments, the control is a positive control sample obtained from a patient diagnosed with CDSRR. In still other embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients with known prognosis or outcome, or a group of samples representing baseline or normal values).

The difference between the test sample and the control may be increased or conversely decreased. The difference may be a qualitative difference or a quantitative difference, e.g. a statistically significant difference. In some examples, the difference is an increase or decrease of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500%, relative to the control.

DNA (deoxyribonucleic acid): DNA is a long-chain polymer that contains genetic material of most organisms (some viruses have genes that contain ribonucleic acids (RNAs)). The repeat units in the DNA polymer are four different nucleotides, each comprising one of the following four bases bound to deoxyribose with a phosphate group attached: adenine (A), guanine (G), cytosine (C) and thymine (T). A triple nucleotide (known as a codon) encodes each amino acid in a polypeptide, or encodes a stop signal. The term codon is also used for a corresponding (and complementary) sequence of three nucleotides in an mRNA into which a DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intended to include the inverse complement of the DNA molecule. Unless the context requires single-stranded, a DNA molecule, while written to describe single-stranded, comprises both strands of a double-stranded DNA molecule. Thus, reference to a nucleic acid molecule encoding a particular protein or fragment thereof includes the sense strand and its inverse. For example, it is suitable to generate probes or primers from the reverse complement of the disclosed nucleic acid molecules.

Expression: transcription or translation of nucleic acid sequences. For example, a coding nucleic acid sequence (such as a gene) may be expressed when its DNA is transcribed into RNA or RNA fragments, which in some instances are processed into mRNA. A coding nucleic acid sequence (such as a gene) may also be expressed when its mRNA is translated into an amino acid sequence (such as a protein or protein fragment). In a particular example, the heterologous gene is expressed when it is transcribed into RNA. In another example, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. Modulation of expression may include controlling transcription, translation, RNA transport and processing, degradation of intermediate molecules (such as mRNA), or by their activation, inactivation, compartmentalization, or degradation after production of a particular protein molecule.

Expression control sequence: a nucleic acid sequence that modulates expression of a heterologous nucleic acid sequence operably linked thereto. Expression control sequences are operably linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription as well as translation (as appropriate) of the nucleic acid sequence. Thus, the expression control sequence may comprise an appropriate promoter, enhancer, transcription terminator, start codon (ATG) in front of the protein encoding gene, splicing signal of the intron, maintenance of the correct reading frame for the gene to allow correct translation of mRNA, and stop codon. The term "control sequences" is intended to encompass at least components whose presence can affect expression, and may also encompass additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. The expression control sequence may comprise a promoter.

Gene: a nucleic acid sequence, typically a DNA sequence, comprising the control and coding sequences necessary for transcription of RNA (whether mRNA or otherwise). For example, a gene may comprise a promoter, one or more enhancers or silencers, a nucleic acid sequence encoding an RNA and/or polypeptide, a downstream regulatory sequence, and possibly other nucleic acid sequences involved in regulating mRNA expression.

As is well known in the art, most eukaryotic genes contain both exons and introns. The term "exon" refers to a nucleic acid sequence present in genomic DNA that is predicted bioinformatically and/or experimentally demonstrated to contribute a contiguous sequence to a mature mRNA transcript. The term "intron" refers to a nucleic acid sequence present in genomic DNA that is predicted and/or verified to not contribute to a mature mRNA transcript, but rather to be "spliced out" during processing of the transcript.

Gene therapy: introducing a heterologous nucleic acid molecule into one or more recipient cells, wherein expression of the heterologous nucleic acid in the recipient cells affects the function of the cells and produces a therapeutic effect in the subject. For example, a heterologous nucleic acid molecule may encode a protein that affects the function of the recipient cell.

Hybridization: hybridization assays for characterizing nucleic acids having a certain level of identity to the nucleic acid sequences provided herein are well known in the art; see, for example Sambrook,Russell"Molecular Cloning,A Laboratory Manual",Cold Spring Harbor Laboratory,N.Y.(2001);Ausubel,"Current Protocols in Molecular Biology",Green Publishing Associates and Wiley Interscience,N.Y.(1989)., the term "hybridization" or "hybridization" as used herein may relate to hybridization under stringent or non-stringent conditions. The hybridization conditions can be established according to conventional protocols described, for example, in Sambrook (2001) loc.cit., ausubel (1989) loc.cit., or Higgins and Hames(Eds.)"Nucleic acid hybridization,a practical approach"IRL Press Oxford,Washington D.C.,(1985). The setting of the conditions is well within the purview of the skilled person and can be determined according to the protocols described in the art. Thus, detection of hybridization sequences typically requires stringent hybridization and wash conditions, such as, for example, conditions from 0.1 XSSC, 0.1% SDS (at 65 ℃) or 2 XSSC, 60 ℃, 0.1% SDS to, for example, 6 XSSC, 1% SDS (at 65 ℃). It is well known that the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions.

Separating: an "isolated" biological component (such as a nucleic acid molecule, protein, virus, or cell) has been substantially isolated or purified from cells or tissues of an organism in which the component naturally resides or other biological components of the organism itself, such as other chromosomal and extra-chromosomal DNA and RNA, proteins, and cells. Nucleic acid molecules and proteins that have been "isolated" include those purified by standard purification methods. The term also includes nucleic acid molecules and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acid molecules and proteins.

Nucleic acid molecules: a polymerized form of nucleotides, which may include the sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the foregoing. Nucleotide refers to ribonucleotides, deoxynucleotides or modified forms of either nucleotide. The term "nucleic acid molecule" as used herein is synonymous with "nucleic acid" and "polynucleotide". Unless otherwise indicated, nucleic acid molecules are typically at least 10 bases in length. The term includes both single-stranded and double-stranded forms of DNA. Polynucleotides may include one or both of naturally occurring nucleotides and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. "cDNA" refers to DNA that is complementary or identical to mRNA in single-or double-stranded form. "coding" refers to the inherent properties of a particular nucleotide sequence (such as a gene, cDNA, or mRNA) in a polynucleotide for use as a template in the synthesis of other polymers and macromolecules in biological processes having defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences, and the biological properties resulting therefrom.

Operatively connected to: the first nucleic acid sequence is operably linked to the second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, if necessary, join two protein coding regions in the same reading frame.

A pharmaceutically acceptable carrier: the pharmaceutically acceptable carriers used are conventional .Remington:The Science and Practice of Pharmacy,22^nd ed.,London,UK:Pharmaceutical Press,2013, describing compositions and formulations suitable for drug delivery with the disclosed carriers.

In general, the nature of the carrier will depend on the particular mode of administration employed. For example, parenteral formulations typically comprise injectable fluids, including pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like as vehicles. For solid compositions (e.g., in powder, pellet, tablet or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to the bio-neutral carrier, the pharmaceutical composition to be administered (such as a carrier composition) may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents, and the like, for example sodium acetate or sorbitan monolaurate. In particular embodiments, a carrier suitable for administration to a subject may be sterile and/or suspended or otherwise contained in a unit dosage form containing one or more measured doses of a composition suitable for inducing a desired immune response. It may also be accompanied by a drug for therapeutic purposes. The unit dosage form may be, for example, in a sealed vial containing sterile contents, or in a syringe injected into a subject, or lyophilized for subsequent dissolution and administration, or in solid or controlled release dosage form.

And (3) purifying: the term "purified" does not require absolute purity; instead, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein (such as KCVN protein) is more enriched in its natural environment within the cell than the peptide or than the protein. In one embodiment, the formulation is purified such that the protein comprises at least 50% of the total protein content of the formulation.

Polypeptide: any amino acid chain, independent of length or post-translational modification (such as glycosylation or phosphorylation). "polypeptide" applies to amino acid polymers, including naturally occurring amino acid polymers, and non-naturally occurring amino acid polymers in which one or more amino acid residues are non-natural amino acids (e.g., artificial chemical mimics of the corresponding naturally occurring amino acid). "residue" refers to an amino acid or amino acid mimetic that is incorporated into a polypeptide by an amide bond or amide bond mimetic. The polypeptide has an amino terminus (N-terminus) and a carboxy terminus (C-terminus). "polypeptide" is used interchangeably with peptide or protein and is used herein to refer to a polymer of amino acid residues.

Preventing, treating or ameliorating a disease: "preventing" a disease (such as a retinal disorder) refers to inhibiting the overall progression of the disease. "treatment" refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop. "ameliorating" refers to reducing the amount or severity of a sign or symptom of a disease.

Promoter: a DNA region that directs/initiates transcription of a nucleic acid (e.g., gene). The promoter comprises the necessary nucleic acid sequence in the vicinity of the transcription initiation site. Typically, promoters are located near the genes they transcribe. The promoter also optionally contains distal enhancer or repressor elements, which may be located up to several thousand base pairs from the transcription initiation site. Tissue-specific promoters are promoters that direct/initiate transcription primarily in a single type of tissue or cell. For example, a photoreceptor-specific promoter is a promoter that directs/initiates transcription in photoreceptor cells to a much greater extent than in other cell types.

Protein: a biological molecule expressed from a gene or other encoding nucleic acid (e.g., cDNA) and consisting of amino acids.

And (3) purifying: the term "purified" does not require absolute purity; instead, it is intended as a relative term. Thus, for example, purified peptides, proteins, viruses or other active compounds are isolated in whole or in part from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to peptides, proteins, viruses, or other active compounds that are isolated from cells, cell culture media, or other crude preparations and fractionated to remove various components of the original preparation (e.g., proteins, cell debris, and other components).

Recombination: a recombinant nucleic acid molecule refers to a nucleic acid molecule having a sequence that is not naturally occurring (e.g., includes one or more nucleic acid substitutions, deletions, or insertions), and/or having a sequence that is made by the artificial combination of two otherwise isolated sequence fragments. Such artificial combination may be achieved by chemical synthesis or, more commonly, by manual manipulation of isolated nucleic acid fragments (e.g., by genetic engineering techniques).

A recombinant virus is a virus comprising a genome comprising a recombinant nucleic acid molecule. As used herein, "recombinant AAV" refers to an AAV particle in which a recombinant nucleic acid molecule (such as a recombinant nucleic acid molecule encoding kv 8.2) is encapsulated.

A recombinant protein is a sequence that has a non-naturally occurring sequence or has an artificial combination of two otherwise isolated sequence fragments. In various embodiments, the recombinant protein is encoded by a heterologous (e.g., recombinant) nucleic acid that is introduced into the genome of a host cell (such as a bacterial or eukaryotic cell) or a recombinant virus.

Retina: the retina consists of a Retinal Pigment Epithelium (RPE) cell layer and three layers of sensory nerve cells; i.e. (from outside to inside): the outer nuclear layer (comprising rod cells and 15 cone photoreceptor cells), the inner nuclear layer (comprising bipolar cells) and the ganglion cell layer. Retinal disorders or dystrophies can be defined as retinal diseases characterized by a gradual loss of photoreceptor cells and concomitant vision loss. The retinal disorder or malnutrition may be a hereditary retinal disorder or malnutrition.

Sequence identity: identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed as identity or similarity between the sequences. Sequence identity can be measured in terms of percent identity; the higher the percentage, the more consistent the sequence. Sequence similarity can be measured as a percentage of similarity (which allows for conservative amino acid substitutions); the higher the percentage, the more similar the sequences. Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity/similarity when aligned using standard methods. Such homology is more pronounced when the orthologous (orthologous) proteins or cdnas are derived from more closely related species such as human and mouse sequences, as compared to more closely related species such as human and caenorhabditis elegans (c.elegans) sequences.

Sequence alignment methods for comparison are well known in the art. Various procedures and alignment algorithms are described in ：Smith&Waterman,Adv.Appl.Math.2:482,1981;Needleman&Wunsch,J.Mol.Biol.48:443,1970;Pearson&Lipman,Proc.Natl.Acad.Sci.USA 85:2444,1988;Higgins&Sharp,Gene,73:237-44,1988;Higgins&Sharp,CABIOS 5:151-3,1989;Corpet et al.,Nuc.Acids Res.16:10881-90,1988;Huang et al.Computer Appls.in the Biosciences 8,155-65,1992; and Pearson et al, meth.mol.Bio.24:307-31, 1994. Altschul et al, J.mol. Biol.215:403-10,1990, present detailed considerations for sequence alignment methods and homology calculations.

NCBI Basic Local Alignment Search Tools (BLAST) (Altschul et al, J.mol. Biol.215:403-10, 1990) are available from a variety of sources, including National Center for Biological Information (NCBI) and the Internet, for use in connection with sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found on the NCBI website.

As used herein, reference to "at least 90% identity" refers to "at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to a particular reference sequence.

The subject: living multicellular vertebrate organisms, including human and non-human mammalian species.

Therapeutically effective amount of: an amount of an agent, such as the disclosed recombinant AAV vector encoding KCVN2, sufficient to prevent, treat (including prophylactic treatment), reduce and/or ameliorate symptoms and/or potential foci of a disorder or disease, e.g., prevent, inhibit and/or treat a retinal disorder. For example, this may be the amount of recombinant AAV vector encoding the novel KCVN2 gene as described herein that produces a sufficient amount of KCVN2 to restore photoreceptor function.

In one example, the desired response is to restore photoreceptor function (e.g., as measured by Electroretinogram (ERG) recording) in a subject, such as a subject with CDSRR. For an effective method, ERG recordings (a-wave, b-wave, c-wave) need not be completely restored to ERG recordings of normal healthy subjects without CDSRR. For example, administration of a therapeutically effective amount of a vector disclosed herein (such as a vector encoding KCVN) can increase photopic or scotopic ERG b waves by a desired amount, e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 100% or more, as compared to a suitable control.

It will be appreciated that multiple administrations of the therapeutic agent may be required in order to obtain a therapeutic response to a disease or condition. Thus, a therapeutically effective amount includes a partial dose that, in combination with a previous or subsequent administration, contributes to achieving a therapeutic effect in a patient. For example, during treatment, a therapeutically effective amount of the agent may be administered in a single dose or in multiple doses, e.g., daily. However, a therapeutically effective amount may depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. The unit dosage form of the medicament may be packaged in a therapeutic amount or in multiples of a therapeutic amount, for example, in a vial (e.g., with a pierceable cap) or syringe with a sterile component.

And (3) a carrier: a vector is a nucleic acid molecule that allows insertion of an exogenous nucleic acid without disrupting the ability of the vector to replicate and/or integrate in a host cell. A vector may comprise a nucleic acid sequence, such as an origin of replication, that allows it to replicate in a host cell. The vector may also contain one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of an inserted gene or genes. In some embodiments herein, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the vector is a gamma retrovirus vector, a lentiviral vector, or an adenovirus vector.

Novel KCVN gene

A nucleic acid molecule encoding a protein having kv8.2 activity is provided, the nucleic acid molecule comprising a nucleotide sequence as shown in SEQ ID No.2 or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

A nucleic acid molecule is provided that hybridizes under stringent conditions to the nucleotide sequence set forth in SEQ ID No.2 or to a complementary strand thereof having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

A nucleic acid molecule is provided which, due to the genetic code, degenerates into the nucleotide sequence shown as SEQ ID No.2 or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

As discussed in example 1, the nucleotide sequence encoding kv8.2 was codon optimized for improved expression. An exemplary optimized KCVN sequence is provided as SEQ ID NO. 2.

Disclosed herein are variants of KCVN2 genes having increased expression relative to corresponding native human KCVN 2. SEQ ID NO. 2 has improved therapeutic properties, including improved photoreceptor therapeutic properties, compared to the unmodified KCVN2 gene (including the human wild-type KCVN gene as disclosed in SEQ ID NO. 1, the coding region of which is disclosed as SEQ ID NO. 12). Improved properties of the disclosed KCVN variants include, but are not limited to, increased protein synthesis, more stable mRNA, increased translational elongation, and/or improved pharmacokinetic properties. Improved properties may include stable transgene and protein expression, enhanced recovery, and improved visual function.

A variant of KCVN polynucleotide may be defined as any variant of SEQ ID NO.2, including naturally occurring variants in the nucleic acid sequence. A variant may be defined as having at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO.2, wherein the polypeptide translated from the variant sequence retains its function. A variant may be defined as having at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO.2, wherein a polypeptide translated from variant sequence SEQ ID NO. 11 has the ability to rescue photoreceptor function. In certain variations, the variant is a codon-optimized version of the coding sequence.

Expression constructs contemplated by the present disclosure can rescue cone photoreceptor function. Rescue of the cone photoreceptor function may be defined as restoring at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the cone photoreceptor function. Cone cell photoreceptor function may be analyzed by any suitable standard technique known to those skilled in the art, such as Electroretinogram (ERG) analysis of retinal responses. Rod cell photoreceptor function may be analyzed by any suitable standard technique known to those skilled in the art (e.g., ERG analysis by retinal reaction).

Rescuing photoreceptor function may also be defined as extending photoreceptor survival. Extending the survival of a photoreceptor may be defined as extending the time that the photoreceptor (e.g., cone cell photoreceptor and/or rod cell photoreceptor) is functional or present by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more than 100% when compared to a photoreceptor affected by malnutrition. Examples of prolonging photoreceptor survival also include improving ERG activity or slowing the loss of ERG activity, improving retinal sensitivity, or slowing/preventing progressive loss of retinal sensitivity, slowing or preventing photoreceptor cell loss, slowing/preventing thinning of the outer retinal layer, improving vision, or slowing/preventing vision loss.

The expression construct may comprise one or more transcriptional control units operably linked to the KCVN gene. In one variant, the KCVN gene is SEQ ID NO. 2. In some variations, the KCVN gene is codon optimized.

Thus, nucleic acid molecules (e.g., cDNA or RNA molecules) encoding kv8.2 are provided, as well as purified forms of kv8.2. In various embodiments, the nucleic acid molecule can be expressed in a host cell (such as a mammalian cell) to produce kv8.2.

The genetic code may be used to construct a variety of functionally equivalent nucleic acid sequences, such as nucleic acids that differ in sequence but encode the same polypeptide sequence.

The nucleic acid molecules disclosed herein may be prepared by any suitable method including: such as cloning of the appropriate sequences or direct chemical synthesis by standard methods. Chemical synthesis produces single stranded oligonucleotides. This can be converted to double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using a single strand as a template.

Exemplary nucleic acids may be prepared by cloning techniques. Examples of suitable cloning and sequencing techniques can be found, for example, in Green and Sambrook(Molecular Cloning:A Laboratory Manual,4^thed.,New York:Cold Spring Harbor Laboratory Press,2012) and Ausubel et al.(Eds.)(Current Protocols in Molecular Biology,New York:John Wiley and Sons,including supplements,2017).

Nucleic acids can also be prepared by amplification methods. Amplification methods include Polymerase Chain Reaction (PCR), ligase Chain Reaction (LCR), transcription-based amplification systems (TAS) and self-sustained sequence replication systems (self-sustained sequence replication system) (3 SR).

The nucleic acid molecules can be expressed in recombinant engineered cells such as bacterial, plant, yeast, insect and mammalian cells. The DNA sequences disclosed herein may be expressed in vitro by DNA transfer into a suitable host cell. The cell may be a prokaryotic cell or a eukaryotic cell. A variety of expression systems are available for protein expression including e.coli (e.coli), other bacterial hosts, yeast and various higher eukaryotic cells such as COS, CHO, heLa and myeloma cell lines for expression of the novel nucleotide sequences disclosed. Methods for stable transfer (meaning that the foreign DNA remains in the host continuously) are known in the art.

Expression of the disclosed nucleic acids can be achieved by operably linking the DNA or cDNA to a promoter (which is constitutive or inducible) and then incorporating into the expression cassette. The promoter may be any promoter of interest, including photoreceptor-specific promoters, such as the Rhodopsin Kinase (RK) promoter (SEQ ID NO: 6). Other exemplary promoters include, but are not limited to, CAG (hybrid CMV early enhancer/chicken b-actin promoter), CBA (chicken b-actin promoter), CBh (hybrid version of CBA promoter), CMV (human cytomegalovirus promoter), CB (regulatory elements consisting of Cytomegalovirus (CMV) immediate early enhancer, chicken b-actin promoter with first intron/exon junction, hybrid chicken b-actin and rabbit b-globulin intron/exon junction), CBSB (regulatory elements comprising CMV immediate enhancer sequence shorter than CB promoter), GRK1 (human G protein coupled receptor kinase 1 promoter), pRLBP (shortened human retinal binding protein 1 (RLBP 1) promoter), hCAR (human cone suppressor protein promoter), pri.7 and PR2.1 (versions of human L-opsin promoter), IRBP (human intersystem photoreceptor (interphotoreceptor) retinoid enhancer, RS/IRBP (irb-enhancer) and human phospho-cleavage protein (human retinal binding protein) promoter), and human phospho-cleavage enzyme (human retinal binding protein 376 promoter).

Optionally, a regulatory element is included in the construct, such as any one or more of the following: KOZAK consensus sequence (SEQ ID NO: 8), woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) (SEQ ID NO: 9) and/or bovine growth hormone polyadenylation (BGH poly (A)) (SEQ ID NO: 10). The cassette may be suitable for replication and integration in prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences that can be used to regulate the expression of the DNA encoding the protein. For example, the expression cassette may comprise appropriate promoters, enhancers, transcriptional and translational terminators, initiation sequences, start codons preceding the protein-encoding gene (i.e., ATG), splicing signals for introns, sequences for maintaining the correct reading frame of the gene to allow for correct translation of the mRNA, and stop codons. The vector may encode a selectable marker, such as a marker encoding resistance to drug (e.g., ampicillin or tetracycline resistance).

In order to obtain high level expression of cloned genes, it is necessary to construct an expression cassette comprising, for example, a strong promoter for directing transcription, a ribosome binding site (e.g., an internal ribosome binding sequence) for translation initiation, and a transcription/translation terminator. For E.coli, this may comprise: promoters (such as T7, trp, lac or lambda promoters), ribosome binding sites, and preferably transcriptional termination signals. For eukaryotic cells, the control sequences may include promoters and/or enhancers derived from, for example, immunoglobulin genes, HTLV, SV40, or cytomegalovirus, as well as polyadenylation sequences, and may also include splice donor and/or acceptor sequences (e.g., CMV and/or HTLV splice acceptors and donor sequences). The cassette may be transferred into the selected host cell by the following well known methods: such as for transformation or electroporation of E.coli and calcium phosphate treatment, for electroporation or lipofection of mammalian cells. The cells transformed by the cassette may be selected by resistance to antibiotics conferred on the genes contained in the cassette, such as amp, GPt, neo and hyg genes.

Nucleic acids encoding polypeptides described herein may be modified without decreasing their biological activity. Some modifications may be made to facilitate the incorporation of cloning, expression or targeting molecules into fusion proteins. Such modifications include, for example, stop codons, sequences for creating restriction sites for convenient positioning, and sequences that add methionine at the amino terminus to provide an initiation site, or additional amino acids such as polyhistidine (polyHis) for aiding in the purification step.

Once expressed, the disclosed kv8.2 can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (typically, see Simpson et al.(Eds.),Basic methods in Protein Purification and Analysis:A Laboratory Manual,New York:Cold Spring Harbor Laboratory Press,2009). the disclosed polypeptide need not be 100% pure if to be used in therapy, the polypeptide should be substantially free of endotoxin once partially purified or to achieve the desired homogeneity.

Recombinant vector and gene therapy application

In current practice, gene therapy means the functional replacement of a dysfunctional gene that does not produce a functional protein with a wild-type copy of the restored function. Gene therapy is a promising approach to the treatment of hereditary and common complex retinal disorders, and preclinical and clinical studies have validated the use of adeno-associated viral vectors (AAV) as a safe and effective gene delivery vehicle. AAV-mediated gene replacement therapies have been implemented in different tissues and systems (including liver, muscle, blood cells, and retina).

Various animal models of genetic photoreceptor degeneration have been successfully treated by gene supplementation therapy, and to date, visual rescue has been achieved at morphological, functional and behavioral levels. There are 43 ongoing or completed clinical trials using AAV delivery systems as a means to correct genetic defects in different types of hereditary retinal disorders (clinical techniques gov, search completed in 11/16/2020). Additional five clinical trials (three are ongoing and two have been completed) are also in use or AAV has been used to provide therapies for age-related macular degeneration.

AAV vectors have proven to be an acceptable delivery method for photoreceptor cell targeting in terms of efficiency and specificity. AAV vectors have been shown to have high affinity for photoreceptor cells while also providing non-toxic, non-pathogenic and low immunogenic characteristics. This demonstrates the successful application of gene therapy in models of retinal disorders and vision loss. Among the different AAV serotypes currently available, the most commonly used serotypes targeted to retinal cells are AAV2/2, AAV2/8, AAV2/9, AAV2/5, AAV2/7m8, AAV2/anc80_l065 serotypes.

Any of the recombinant nucleic acid molecules discussed above may be contained in a vector (such as an AAV vector) for expression in a cell or subject.

The nucleic acid sequences disclosed herein can be used in production vectors (such as rAAV vectors), as well as in antisense delivery vectors, gene therapy vectors, or vaccine vectors. In certain embodiments, the present disclosure provides gene delivery vectors and host cells comprising the nucleic acid sequences disclosed herein. In some embodiments, the selected vector may be delivered to the subject by any suitable method, including intravenous injection, ex vivo transduction, transfection, electroporation, liposome delivery, membrane fusion techniques, high speed DNA coated pellets, viral infection, or protoplast fusion, to introduce the transgene into the subject.

In certain embodiments, the disclosure relates to viral particles, e.g., capsids, comprising the KCVN nucleic acid sequence SED ID NO.2 disclosed herein. Viral particles, capsids, and recombinant vectors can be used to deliver nucleic acid sequences to target cells. Nucleic acids can be readily used in a variety of vector systems, capsids, and host cells. In certain embodiments, the nucleic acid is in a vector contained within a capsid comprising cap proteins (including AAV capsid proteins vp1, vp2, vp 3) and hypervariable regions.

In certain embodiments, KCVN nucleic acid sequences may be part of any genetic element (vector) that can be delivered to a host cell, such as naked DNA, plasmids, phages, transposons, cosmids, episomes, proteins in non-viral delivery vehicles (e.g., lipid based vectors), viruses, and the like, that can transfer sequences carried thereon.

In certain embodiments, the vector may be a lentiviral-based (comprising a lentiviral gene or sequence) vector, e.g., having a nucleic acid sequence derived from the VSVG or GP64 pseudotype, or both. In certain embodiments, a nucleic acid sequence derived from a VSVG or GP64 pseudotype may be at least one or two or more genes or gene fragments having greater than 1000, 500, 400, 300, 200, 100, 50, or 25 consecutive nucleotides or nucleotide sequences with greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the gene or fragment.

In some embodiments, the nucleic acid and promoter sequences disclosed herein are useful in the production of AAV vectors. AAV belongs to the parvoviridae (Parvoviridae) and dependovirus (Dependovirus). AAV is a small non-enveloped virus that encapsulates a linear single-stranded DNA genome. Both the sense and antisense strands of AAV DNA are packaged into AAV capsids at the same frequency. AAV genomes are characterized by two Inverted Terminal Repeats (ITRs) flanking two Open Reading Frames (ORFs). In the AAV2 genome, the first 125 nucleotides of an ITR, for example, are palindromic sequences that fold upon themselves to maximize base pairing and form a T-shaped hairpin structure. The other 20 bases of the ITR (called D sequence) remain unpaired. ITR is a cis-acting sequence important for AAV DNA replication; ITR is the origin of replication and serves as a primer for the synthesis of the second strand by DNA polymerase. Double-stranded DNA (known as replicative monomers) formed during this synthesis is used for the second round of self-priming replication (self-priming replication) and forms replicative dimers. These double-stranded intermediates are processed by a strand displacement mechanism to produce single-stranded DNA for encapsulation and double-stranded DNA for transcription. Located within the ITR are the Rep binding element and a terminal melting site (terminal resolution site, TRS). These features are used by the viral regulatory protein Rep to process double stranded intermediates during AAV replication. In addition to its role in AAV replication, ITR is also essential for AAV genome packaging, transcription, negative regulation under unlicensed conditions, and site-specific integration (Daya and Berns, clin Microbiol Rev 21 (4): 583-593, 2008).

AAV vectors typically contain a transgene expression cassette between ITRs that replaces the rep and cap genes. Vector particles are generated by co-transfecting cells with a plasmid containing the vector genome and a packaging/helper construct that expresses rep and cap proteins in trans. During infection, the AAV vector genome enters the nucleus and can persist in a variety of molecular states. One common outcome is the conversion of the AAV genome into a double-stranded circular episome by second strand synthesis or complementary strand pairing.

In the context of AAV vectors, the disclosed vectors typically have a recombinant genome comprising the following structure:

(5 'AAV-ITR) - (promoter) - (transgene) - (3' AAV-ITR)

As described above, these recombinant AAV vectors comprise a transgene expression cassette between ITRs that replaces the rep and cap genes. For example, vector particles are produced by co-transfecting cells with a plasmid comprising a recombinant vector genome and a packaging/helper construct that expresses rep and cap proteins in trans.

The transgene may be flanked by regulatory sequences, such as 5'kozak sequences and/or 3' polyadenylation signals.

AAV ITRs and other selected AAV components described herein can be readily selected from any AAV serotype, including, but not limited to AAV1、AAV2、AAV2-QuadyF、AAV2.7m8、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV8(Y733F)、AAV9、Anc80、AAV7m8、AAVrh10、AAV-PHP.eB、AAV-PHP.S、AAV-DJ、AAV-DJ/8、AAV2.GL、AAV2.NN、AAVAnc80_L065 and any functional variants thereof. These ITRs or other AAV components can be readily isolated from AAV serotypes using techniques available to those skilled in the art. Such AAV may be isolated or obtained from an academic, commercial, or public source (e.g., american type culture collection (THE AMERICAN TYPE Culture Collection), manassas, va.). Alternatively, AAV sequences may be obtained by synthetic means or other suitable means by reference to published sequences, such as in a document or database (such as GenBank, pubMed, etc.). It is to be understood that the present disclosure encompasses AAV genomes using other serotypes that may not have been identified or characterized.

In some embodiments, the recombinant AAV vector genome may have a photoreceptor-specific promoter, such as Rhodopsin Kinase (RK) or any modification thereof. The recombinant AAV vector genome may have any promoter known in the art, including those disclosed herein and/or those disclosed in Kaneshiro,K.,Wu,Z.,Li,T.,Sieving,P.,&Colosi,P.(2011);Evaluation of Viral and Human Retinal Promoters in AA V8 Vectors.Investigative Ophthalmology&Visual Science,52(14),491.

AAV is one of the most commonly used viruses currently used for gene therapy. Although AAV infects humans and some other primate species, it is known to not cause disease and elicit a very mild immune response. Gene therapy vectors utilizing AAV can infect dividing and resting cells and persist in an extrachromosomal state without integration into the host cell's genome. Because of the advantageous features of AAV, the present disclosure contemplates the use of AAV with the recombinant nucleic acid molecules and methods disclosed herein.

AAV has a number of desirable features for use in gene therapy vectors, including binding and entry into target cells, the ability to enter the nucleus, the ability to be expressed in the nucleus for extended periods of time, and low toxicity. However, the small size of the AAV genome limits the size of heterologous DNA that can be incorporated. To minimize this problem, AAV vectors have been constructed that do not encode Rep and Integration Efficiency Elements (IEEs). ITRs are retained because they are cis signals required for encapsulation (Daya and Berns, clin Microbiol Rev (4): 583-593, 2008).

Methods for producing rAAV suitable for Gene therapy are known (see, e.g., U.S. patent application nos. 2012/0100606, 2012/0135515, 2011/0229971 and 2013/0074248; and Ghosh et al, gene ter 13 (4): 321-329, 2006), and can be used with the recombinant nucleic acid molecules and methods disclosed herein.

In some embodiments, the nucleic acids disclosed herein are part of an expression cassette or transgene. See, for example, U.S. patent application number 20150139953. Expression cassettes consist of transgene and regulatory sequences such as promoters and 5 'and 3' aav Inverted Terminal Repeats (ITRs). In a desirable embodiment, ITRs of AAV serotypes 2 or 8 are used. However, ITRs from other suitable serotypes may be selected. The expression cassette is typically packaged into a capsid protein and delivered to a selected host cell.

In some embodiments, the disclosure provides methods of producing a recombinant adeno-associated virus (AAV) or a portion thereof having an AAV serotype capsid. Such methods include culturing a host cell comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) serotype capsid protein; a functional rep gene; an expression cassette consisting of an AAV Inverted Terminal Repeat (ITR) and a transgene; and sufficient helper functions to allow encapsulation of the expression cassette into AAV capsid proteins. See, for example, U.S. patent application publication 20150139953.

In some embodiments, the disclosure relates to recombinant vectors comprising photoreceptor-specific promoter nucleic acid sequences operably combined with transgenes. A transgene is a nucleic acid sequence heterologous to the vector sequence flanking the transgene, which encodes kv8.2 as disclosed herein and optionally one or more additional proteins of interest. The nucleic acid coding sequence is operably linked to the regulatory component in a manner that allows for the transcription, translation, and/or expression of the transgene in the host cell.

The expression cassette may be carried in any suitable vector, such as a plasmid, that is delivered to the host cell. Plasmids useful in the present disclosure may be engineered such that they are suitable for replication and optionally integration in prokaryotic cells, mammalian cells, or both. These plasmids (or other vectors carrying 5'aav ITR heterologous molecules-3' ITRs) contain sequences that allow the expression cassette to replicate in eukaryotes and/or prokaryotes, as well as selection markers for these systems. Preferably, the molecule carrying the expression cassette is transfected into a cell, and the molecule may be transiently present in the cell. Alternatively, the expression cassette (carrying the 5'aav ITR heterologous molecule-3' ITR) may be stably integrated into the genome of the host cell by chromosome or as an episome. In certain embodiments, the expression cassette may be present in multiple copies, optionally in a head-to-head, head-to-tail, or tail-to-head concatemer (concatamer). Suitable transfection techniques are known and can be readily used to deliver the expression cassette to a host cell.

In general, when delivering vectors comprising expression cassettes by transfection, the relative amounts of vector and vector DNA into the host cell may be adjusted in view of factors such as the vector selected, the method of delivery, and the host cell selected. In addition to the expression cassette, the host cell comprises sequences that drive expression of the AAV capsid protein in the host cell, and rep sequences of the same serotype, or of a cross-complementary serotype, as the serotype of the AAV ITRs present in the expression cassette. Although the molecule providing rep and cap may be transiently present in the host cell (i.e., by transfection), preferably one or both of the rep and cap proteins and the promoter controlling their expression are stably expressed in the host cell, for example, as an episome or by integration into the chromosome of the host cell.

Introduction of the vector into the host cell may be accomplished by any means known in the art or as disclosed above, including transfection, infection, electroporation, liposome delivery, membrane fusion techniques, high speed DNA coated pellets, viral infection, and protoplast fusion, among others. One or more adenovirus genes may be stably integrated into the genome of the host cell, stably expressed as episomes, or transiently expressed. The gene products may all be transiently expressed (episomally or stably integrated), or some may be stably expressed while others are transiently expressed. In addition, the promoter of each adenovirus gene may be independently selected from a constitutive promoter, an inducible promoter, or a native adenovirus promoter. For example, a promoter may be regulated by a particular physiological state of an organism or cell (i.e., by a differentiated state or in replicating or dormant cells) or by exogenously added factors.

AAV technology may be suitable for use in these and other viral vector systems for in vitro, ex vivo, or in vivo gene delivery. In certain embodiments, the present disclosure contemplates the use of the nucleic acids and vectors disclosed herein in various rAAV and non-rAAV vector systems. Such vector systems may include, for example, lentivirus, retrovirus, poxvirus, vaccinia virus, and adenovirus systems, and the like.

In some embodiments, it is contemplated that the viral particles, nucleic acids, and vectors disclosed herein can be used for a variety of purposes, including for delivering therapeutic molecules for gene expression of therapeutic proteins.

The therapeutic proteins encoded by the nucleic acids reported herein (e.g., in operable combination with a promoter) include those useful for treating retinal disorders.

In some embodiments, a method of restoring retinal function in a subject having CDSRR is disclosed. The method comprises administering to the subject a therapeutically effective amount of a vector (such as an AAV vector, a lentiviral vector, or a retroviral vector) comprising a KCVN nucleic acid sequence as described herein. In some embodiments, the subject is a subject having a retinal disorder (such as CDSRR).

In one variant, we delivered the novel KCVN gene in AAV2/8 serotypes, which exhibit up to 100-fold higher transduction capacity compared to some other known capsids. In the retina, AAV2/8 appears to be a more potent vector than AAV2/2 and AAV 2/5; AAV2/8 provides faster onset and stronger and higher transgene expression, particularly in photoreceptors.

In some embodiments, the disclosure relates to gene transfer methods for treatment of CDSRR using adeno-associated virus serotype 8 (AAV 2/8) vectors carrying codon-optimized human KCVN (potassium voltage-gated channel modifier subfamily V member 2). In another variant, the disclosure relates to a gene transfer method for treating CDSRR using an adeno-associated virus serotype 8 (AAV 2/8) vector carrying codon-optimized human KCVN under expression of a Rhodopsin Kinase (RK) promoter. The vector may be referred to herein as AAV2/8.Rk. Hkcvn2.

Delivery of the vector encoding the transgene may be, for example, by direct administration to a subject (e.g., by subretinal injection of the vector). In another example, delivery of the vector may be, for example, by injecting the vector into one or both eyes of a patient in any disease state in the macular and/or foveal area.

In another variation, delivery of the vector encoding the transgene may be by, for example, direct administration to the subject (e.g., by subretinal injection of the vector). In another example, delivery of the vector may be, for example, by injecting the vector into one or both eyes of a patient in any disease state in the macular and/or foveal area. In this variant, AAV2/8.rk.hkcvn2 can be delivered in neutral phosphate buffered saline with Pluronic F68 (0.001%), 0.10ml, dose range 5E9 to 5E11 vector genome.

In general, delivery may be by injection to direct retinal, subretinal or intravitreal delivery of the nucleic acids disclosed herein (such as vectors). In one example, delivery may be by injection into the retina, subretinal space, or intravitreal space.

Thus, we also provide a method of treating or preventing malnutrition (particularly CDSRR) in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a nucleic acid disclosed herein (such as a vector) by direct retinal, subretinal or intravitreal injection.

In a related aspect, the invention provides the use of a nucleic acid disclosed herein (such as a vector) in a method of treating or preventing a retinal disorder (such as malnutrition, particularly CDSRR) by administering the vector to a patient by direct retinal, subretinal, or intravitreal injection.

Furthermore, we provide the use of a nucleic acid disclosed herein (such as a vector) in the manufacture of a medicament for the treatment or prophylaxis of a retinal disorder (such as malnutrition, particularly CDSRR) by direct retinal, subretinal or intravitreal injection.

The invention also provides a nucleic acid disclosed herein (such as a vector) for use in treating a retinal disorder (dystrophy, particularly CDSRR), wherein the vector is directly applied to the retina, subretinal space, or intravitreal space.

The nucleic acids disclosed herein (such as vectors) are typically administered by direct retinal or subretinal injection. This includes delivery directly to cone cell photoreceptor cells and/or rod cell photoreceptor cells.

Optionally, the compositions of the present disclosure may comprise other pharmaceutically acceptable excipients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The recombinant viral particles, capsids, or vectors are administered in an amount sufficient to transfect the cells and provide sufficient levels of gene transfer and expression to provide therapeutic benefit without undue adverse effects, or to produce a physiologically acceptable effect, as can be determined by one of skill in the medical arts. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the desired organ (e.g., eye), oral, inhalation, nasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parental (parental) routes of administration. The routes of administration may be combined, if desired.

The dose of recombinant viral particles, capsids or vectors will depend primarily on, for example, the condition being treated, the age, weight and health of the patient, and thus may vary from patient to patient. For example, a therapeutically effective human dose of viral vector is typically in the range of about 0.1ml to about 100ml of a solution containing a concentration of about 1x 10 ⁹ to 1x 10 ¹⁶ genomic viral vector.

The recombinant viral vectors of the present disclosure provide an effective gene transfer vehicle that can deliver a protein of choice to a host cell of choice in vivo or ex vivo, even in the event that the organism has neutralizing antibodies to the protein of choice. In one embodiment, the vector and cells disclosed herein are mixed ex vivo; culturing the infected cells using conventional methods; and re-infusing the transduced cells into the patient.

Turning to fig. 1, a disclosed vector. The vector for delivering the codon optimized KCVN gene of disclosed SEQ ID NO. 2 may have one or more of the following elements:

Serotype AAV2/8 vectors

Rhodopsin Kinase (RK) promoter (SEQ ID NO: 6)

Codon-optimized KCVN gene of SEQ ID NO. 2

Regulatory elements, including KOZAK consensus sequence between the promoter and KCNV gene (SEQ ID NO: 8), woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) after KCNV gene (SED ID NO: 9), and bovine growth hormone polyadenylation (BGH poly (A)) downstream of WPRE (SEQ ID NO: 10) signals. The disclosed vector may have the nucleotide sequence of SEQ ID NO. 3.

Turning to fig. 2, a second disclosed carrier. The vector for delivering the codon optimized KCVN gene of disclosed SEQ ID NO. 2 may have one or more of the following elements:

AAV vectors

Rhodopsin Kinase (RK) promoter (SEQ ID NO: 6)

Codon-optimized KCVN gene of SEQ ID NO. 2

EGFP enhanced green fluorescent protein

Regulatory elements, including KOZAK consensus sequence (SEQ ID NO: 8) between the promoter and KCNV gene (SEQ ID NO: 2), woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) (SEQ ID NO: 9) after the KCNV gene (SEQ ID NO: 2), and bovine growth hormone polyadenylation (BGH poly (A)) downstream of WPRE (SEQ ID NO: 10) signals.

Turning to fig. 3, a third disclosed vector. The vector for delivering the codon optimized KCVN gene of disclosed SEQ ID NO. 2 may have one or more of the following elements:

AAV vectors

Rhodopsin Kinase (RK) promoter (SEQ ID NO: 6)

Codon-optimized KCVN gene of SEQ ID NO. 2

EGFP enhanced green fluorescent protein

Regulatory elements, including KOZAK consensus sequence (SEQ ID NO: 8) between the promoter and KCNV gene (SEQ ID NO: 2), CMV promoter (SEQ ID NO: 7), woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) (SEQ ID NO: 9) after the KCNV gene, and bovine growth hormone polyadenylation (BGH poly (A)) signal (SEQ ID NO: 10) downstream of the WPRE (SEQ ID NO: 9).

Turning to fig. 4, a fourth disclosed vector. The vector for delivering the codon optimized KCVN gene of disclosed SEQ ID NO. 2 may have one or more of the following elements:

AAV vectors

Rhodopsin Kinase (RK) promoter (SEQ ID NO: 6)

Codon-optimized KCVN gene of SEQ ID NO. 2

EGFP enhanced green fluorescent protein

Regulatory elements, including KOZAK consensus sequence (SEQ ID NO: 8) between the promoter and KCNV gene (SEQ ID NO: 2), CMV promoter (SEQ ID NO: 7), lac promoter, woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) (SEQ ID NO: 9) after the KCNV gene, and bovine growth hormone polyadenylation (BGH poly (A)) signal (SEQ ID NO: 10) downstream of WPRE (SEQ ID NO: 9).

AAV serotype selection

In our exemplary disclosure, we have selected AAV2/8 serotypes that exhibit up to 100-fold higher transduction capacity compared to some other known capsids. We also selected the Anc80 serotype. In the retina, AAV2/8 appears to be a more potent vector than AAV2/2 and AAV 2/5; it provides for faster onset and stronger higher transgene expression, particularly in photoreceptors. However, the Anc80 serotype was also used instead of AAV2/8.

Promoters

The necessary levels of KCNV gene expression to provide the rescue of retinal function are not clear, but we believe that KCVN2 expression may be limited to cone and rod photoreceptor cells. Since KCNV gene encodes a channel protein, the general expression in different retinal cells may have unexpected consequences. For our therapeutic construct, we decided to use the published Rhodopsin Kinase (RK) promoter, as it only limits transgene expression to photoreceptors.

KCNV2 Gene

The human KCNV gene (NCBI reference sequence: NM-133497.4; ensemble gene ENSG00000168263.9 and transcripts KCNV2-201 ENST00000382082.4) (SEQ ID NO: 1) is the transgene of choice in our construct. KCNV2 the transcript has only one splice variant and consists of two exons. The entire transcript was 2178 base pairs long and the coding sequence (CDS) used as the base in our study was 1638 base pairs long (SEQ ID NO: 12).

Prior to construct design, a codon optimized version of the human KCNV CDS (https:// sg. Idtdna. Com) was generated using the codon optimization tool of integrated DNA Technologies, inc. (INTEGRATED DNA Technologies, IDT). The principle is to correlate codon usage with tRNA production to improve transcript stability and prevent aberrant transcript splicing. The IDT algorithm used provides optimal sequence selection by screening and filtering sequences, thereby reducing complexity and minimizing secondary structure.

Adjusting element

All our transgenic constructs also contained a KOZAK consensus sequence (SEQ ID NO: 8) between the promoter and KCNV gene (SEQ ID NO: 2), a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) (SEQ ID NO: 9) after the KCNV gene (SEQ ID NO: 2), and bovine growth hormone polyadenylation (BGH poly (A)) downstream of the WPRE (SEQ ID NO: 10) signals. KOZAK sequence (SEQ ID NO: 8) plays a role in the initiation of protein translation in eukaryotic mRNA transcripts. WPRE (SEQ ID NO: 9) is a DNA sequence that, when transcribed, produces a tertiary structure that enhances expression. This sequence is used to increase the expression of genes delivered by the viral vector. BGH poly (A) (SEQ ID NO: 10) is a specific termination sequence for protein expression in eukaryotic cells, which results in the formation of a poly (A) tail at the 3' end of the mRNA. In addition, mammalian DNA viruses that replicate in the nucleus utilize cellular polyadenylation mechanisms. Poly (a) tails support the stability, transport and translation of most mrnas.

Preparation of the Carrier

The disclosed vectors may be prepared by standard means known in the art for providing therapeutic vectors. Thus, well established methods of public domain transfection, encapsulation and purification can be used to prepare suitable vector formulations.

Constructs were developed by the following steps:

1. Construct design

The design of the therapeutic construct was considered as follows: what promoters should be used to achieve the desired level of expression, whether the gene should be codon optimized, what polyA sites should be used, and whether any other known regulatory sequences should be used.

2. Construct assembly

The DNA fragment of the final construct was synthesized by Genewiz and then cloned into the pAAV backbone plasmid.

3. Packaging to AAV

Once cloned into the pAAV backbone, our construct was purified and larger plasmid amounts were prepared using commercial kits from Qiagen. Purified plasmids were sent for encapsulation in AAV. AAV production followed standard protocols. A batch of AAV2/8 was prepared using our therapeutic plasmid and stored in dry ice.

Examples

The following examples are provided to illustrate certain specific features and/or embodiments. These examples should not be construed as limiting the disclosure to the particular features or embodiments described.

Example 1

Proof of concept study of selected treatments (PC) -AAV8.RK. HKCNV2 gene therapy reduced abnormal b wave amplitude

Our basic research data is used to guide us in deciding which of the three potential therapeutic products tested will be the product we choose. Based on gene expression data showing an average 8-fold increase in gene expression compared to wild type compared to other products, and based on ERG positive b wave data showing a greater difference and a significant decrease compared to untreated eyes, we selected aav8.Rk. Hkcnv2 vector as our therapeutic product for the following examples. However, it should be understood that other AAV serotypes are also intended to include, but are not limited to, AAV Anc80. The following we provide authentication data.

The protocol for the mouse pilot study (pilot studies) is as follows. See fig. 11.

1. Viral particle dilution and storage

The original stock vector was stored at-80 ℃. The working stock was diluted to 1X 10 ¹² vector genomes (vg)/ml in sterile PBS, aliquoted into 20. Mu.l aliquots in 0.2ml sterile PCR tubes (SSIbo UltraFlux FLAT CAP PCR tubes, catalog number 322-00) and stored at-80 ℃. On the day of injection, the diluted aliquots were thawed on ice (4 ℃) prior to injection. A new aliquot was used per batch of injections per day. During injection, the aliquots were kept on ice. Any remaining carrier suspension in the thawed aliquots was discarded and not used in the study.

2. Subretinal injection

P28-35K 8.2 KO ampholytic mice were placed under general anesthesia and injected intraperitoneally with ketamine (100 mg/kg) and xylazine (20 mg/kg). The pupil was dilated by topical application of 1% topiramate (MYDRIACYL; alcon).

A small incision was made under a surgical microscope through the temporal sclera of the eye with the tip of a 30 gauge needle. A 35 gauge stainless steel beveled or blunt needle mounted to a NanoFil syringe was inserted through the incision and a virus suspension/PBS was injected into the subretinal space using an ultra-micro pump (World Precision Instrument) (SOP # 1.06.08).

Each treated animal received subretinal injection of 1 μl of rAAV2/8-hGRK1-hKCNV2 (1×10 ¹² GC/mL) vector (SEQ ID NO: 3) in one eye (n=10, left or right randomly assigned). The other eye remained uninjected. Control animals received subretinal injection of 1 μl of sterile 1X Phosphate Buffered Solution (PBS) in one eye (n=10, randomly assigned left or right eye) while the other eye remained uninjected as an internal control (n=20).

OCT verification was then performed to check the injection location and range indicated by a large vesicular (bleb (bleb)) retinal detachment.

3. Electroretinogram (ERG)

KCNV2 KO mice were subjected to the scotopic ERG test at specific time intervals (4, 8 and 12 weeks after injection) to determine if the a-wave and b-wave amplitudes improved after treatment.

Mice were dark adapted overnight (at least 8 hours) prior to ERG experiments and then treated only under dark red light. ERG was performed as described above [6]. Mice were anesthetized using isoflurane inhalation anesthesia and pupil dilation was performed with local 1% topiramate (MYDRIACYL; alcon). An artificial lubricant (hydroxypropyl methylcellulose) was applied to the cornea prior to placing the ERG electrodes on each eye to avoid dehydration and promote contact. For reference, a subcutaneous needle electrode was placed at each cheek or along the mandible of the mouse, and a ground electrode was placed subcutaneously above the tail bottom.

ERG recordings were obtained using a single flash intensity series of dark adaptation by presenting a 1ms flash at the following intensities (all in cd-s m ^-2): 0.1, 0.3, 1,3, 10 and 25. The time interval between successive flashes and the number of repeated stimuli (for subsequent averaging) varies according to stimulus intensity; 10s and 4 replicates for 0.1-3 cd-s m ^-2 and 60s and 1 replicate for 10-25 cd-s m ^-2. There is a 60 second interval between each group of flashes.

At a background brightness of 30cd.s m ^-2, photopic ERG was recorded at weeks 4 and 12 post-injection after light adaptation. ERG recordings were obtained using a single flash intensity series by presenting a 1ms flash at the following intensities (all in cd-s m ^-2): 0.3, 1,3, 10 and 25. Each stimulus was repeated 32 times (for subsequent averaging), with a time interval between flashes of 0.5s. There is a 60 second interval between each group of flashes. Maximum amplitude and absolute time of a-wave and b-wave are extracted from ERG reactions (IMPLICIT TIME), see in detail (Collison,F.T.,J.C.Park,G.A.Fishman,E.M.Stone,and J.J.McAnany,Two-color pupillometry in KCNV2 retinopathy.Doc Ophthalmol,2019,139(1),p.11-20.doi:10.1007/s10633-019-09691-w),, which is incorporated herein by reference in its entirety.

4. Optical Coherence Tomography (OCT)

OCT imaging was performed 0-3 days post injection, 2 weeks post injection, and 12 weeks post injection to assess whether treatment improved the total thickness of the retinal layer compared to untreated or sham-treated (SHAM TREATED) eyes.

Mice were anesthetized and pupils dilated as previously described. OCT was performed using a spectral domain optical coherence tomography system (Bioptigen Envisu R SD-OCT system). Retinal layer thickness was measured using Bioptigen and ImageJ software.

5. Histopathology

5.1 Slicing

Eyes were collected from treated mice and PBS-injected mice 12 weeks after injection. Briefly, eyes were fixed in 4% PFA for 1 hour on ice. The cornea and lens were dissected and the eye was then incubated overnight at 4 ℃ in 20% sucrose. The next day, eyes were frozen in the compound at the optimal cutting temperature and stored at-20 ℃ and then sectioned. Retinal sections were collected on super-cold (super-frame) slides (Hurst) and cut at 14 μm using Leica Cryostat CM3050,3050. The eyes were sectioned on the sagittal plane and the sections were collected sequentially on 10 slides. After sectioning, the slides were stored at-20 ℃ for further analysis.

5.2 Immunohistochemistry

The presence of kv8.2 protein was assessed in situ in frozen retinal sections from the corresponding age-treated group, sham-surgically injected, uninjected KCNV-/-and uninjected wild-type retinas using kv8.2 Antibodies (Antibodies Inc, USA, cat. No. 75-435/73-435) according to the protocol published in (Skarnes,W.C.,B.Rosen,A.P.West,M.Koutsourakis,W.Bushell,V.Iyer,A.O.Mujica,M.Thomas,J.Harrow,T.Cox,D.Jackson,J.Severin,P.Biggs,J.Fu,M.Nefedov,P.J.de Jong,A.F.Stewart,and A.Bradley,A conditional knockout resource for the genome-wide study of mouse gene function.Nature,2011,474(7351),p.337-42.doi:10.1038/nature10163). We also evaluated in situ expression of rhodopsin (Rho) (Abcam, cat. No. Ab 3424) and cone inhibitor protein (Arr 3) (Millipore, cat. No. Ab 15282).

6.0 Gene expression by qualitative real-time polymerase chain reaction (qRT-PCR)

Total RNA from retinas of treated mice and sham-treated mice was extracted at 12 weeks post injection using Trizol reagent according to published protocols. And then useFirst strand cDNA synthesis kit (NEB, catalog number E6560S) or QuantiTect reverse transcription kit (Qiagen catalog number 2205311), RNA samples were transcribed into complementary DNA (cDNA) by reverse transcriptase according to the manufacturer' S recommended protocol. The cDNA was then used as a template for qRT-PCR reactions.

The level of expression of KCNV, cone-cell arrestin and rhodopsin was assessed using a Taqman-based assay (Thermo Fisher) and a real-time PCR detection machine (Bio-Rad CFX Connect real-time system).

Statistical analysis of data

By obtaining mean and standard deviation values, mRNA expression levels of retinal layer thickness form OCT imaging, maximum amplitude and absolute time of a-wave and b-wave from ERG response, retinal layer thickness form histology, and KCNV, cone arrestin and rhodopsin according to qRT-PCR from treated, PBS-treated and untreated eyes were compared. Statistical significance was assessed by applying standard t-test/one-way ANOVA.

Example 2

Turning to fig. 5, we illustrate an exemplary method for restoring retinal function. Cone cell dystrophy with a supernormal rod cell response, e.g., due to KCVN gene mutations, is a target condition. In one example, the therapeutic intervention is AAV8.RK. HKCVN2 (SEQ ID NO: 3), in which case it would be KCVN2 disclosed as codon optimized for SEQ ID NO: 2. An exemplary dosing regimen is shown as 0.10ml of 5E9-5E11 vector genome (vg) delivered in one or more subretinal injections. As described herein, the design of the vector specifically targets gene expression in photoreceptor cells, and more specifically in the macular and/or foveal regions of the retina. The expected effect is a decrease in the positive b-wave amplitude, which is associated with an improvement in visual function.

Example 3

Turning to fig. 6, we provide an overview of the concept verification study. Stage I is the basic study of wild type animals. This involves first testing the delivery method and verifying the utility of subretinal injection (denoted as FS 1). Injecting fluid into the subretinal space creates blisters, the temporal and focal separation of the photoreceptors supporting the Retinal Pigment Epithelium (RPE). The timing of bubble dissipation is an indicator of photoreceptor cell function. OCT imaging was used on the day of injection and at 2 and 12 weeks post injection to measure effects. Optical Coherence Tomography (OCT) is a non-invasive imaging test. OCT imaging uses light waves to take a cross-sectional photograph of the retina. This allows visualization of the different layers of the retina, rendering the layers and measuring the layer thickness.

After validating the delivery method, the desired promoter (designated FS 2) targeting the photoreceptor is confirmed. The RK and CMV promoters were compared to determine the ability of each to target photoreceptors. In this case, the effect was measured by histological analysis of the retina twelve (12) weeks after injection.

Functional safety assessment was performed to evaluate the effect of subretinal injection on retinal function (designated FS 3). The effects were measured by Electroretinogram (ERG) recordings at 4, 8 and 12 weeks after injection.

Stage II involves a study of the investigational product selection for kv8.2 KO animals. At this stage, three therapeutic products were compared, each with a different AAV serotype and promoter. These three therapeutic products are, for example, aav8.rk.hjcvn2, anc80.rk.hkcvn2 and aav8.cmv.hkcvn2. The effect on injection safety was measured by OCT imaging on the day of injection, 2 weeks and 12 weeks after injection. The effect on functional assessment was measured by ERG recordings at 4, 8 and 12 weeks after injection. The effect on gene expression was measured by retinal KCVN gene expression analysis 12 weeks after injection.

Turning to fig. 7, we outline the phase III proof of concept study. In PC1, we established the efficacy of the vector expressing the optimized KCVN gene of SEQ ID NO. 2. Treatment protocol as outlined in example 1 above. Controls included non-injected mice, wild-type mice with product injected subretinally. These were compared to Kv8.2 KO animals injected with the therapeutic AAV8.RK. HKCVN2 (e.g., SEQ ID NO: 3). The effects were measured by OCT (at weeks 0, 3 and 12 post-injection), ERG (at weeks 4, 8, 12 post-injection) and KCVN gene expression data (at week 12 post-injection). At PC2, recovery of visual function was assessed to observe the effect of delivering the treatment at higher doses. Here, the therapeutic aav8.rk.hkcvn2 (e.g., SEQ ID NO: 3) was subjected to subretinal injection using two dosage regimens: 2X 10-9 vector genome (vg) and 5X 10-9 vector genome (vg). The control was an uninjected mouse. The effects were measured by OCT (at weeks 0, 3 and 12 post-injection), ERG (at weeks 4, 8, 12 post-injection) and KCVN gene expression data (at week 12 post-injection). At PC3, safety and biodistribution were evaluated. We evaluated the safety and biodistribution of single-sided subretinal injection of AAV8.RK. HKCVN2 (e.g., SEQ ID NO: 3) in Kv8.2 KO animals. Three treatment groups received treatment by subretinal injection: group 1 received PBS vehicle only; group 2 receives the therapeutic at a dose of 1 x 10 pattern 9 vector genome (vg)/eye; and group 3 receives the therapeutic at a dose of 5 x 10 pattern 9 vector genome (vg)/eye. All animals were homozygous kv8.2 KO. Effects were measured by ophthalmic examination, ERG, hematology and clinical chemistry, ocular histopathology and biological distribution of vector genome in major organs. Animals were examined at 4 and 12 weeks post injection.

Overall, the data from these basic studies is used to guide the decision on which three potential therapeutic products to use. Based on the gene expression data, the disclosed SEQ ID NO.2 shows an average 8-fold increase in gene expression compared to wild type KCVN 2. ERG positive b wave data showed a greater difference and a significant decrease compared to untreated eyes and other test products. Thus, AAV8.RK. HKCVN2 vector (SEQ ID NO: 3) was selected as a therapeutic treatment product.

Results of the selection

Functional ERG analysis of treated eyes compared to wild type

Turning to fig. 8, fig. 8 provides a comparison between treated and untreated mice using the disclosed methods, systems and nucleic acid sequences, as discussed in fig. 8. To evaluate the efficacy of treatment with SEQ ID NO. 2 and further with vector SEQ ID NO. 3 to restore photoreceptor function in a subject (e.g., a subject with CDSRR), positive b-wave ERG amplitude was compared between treated (CDSRR animals represented by Kv8.2 animals), untreated (CDSRR animals represented by Kv8.2 animals) and wild-type eyes (wild-type animals). At 4, 8 and 12 weeks post injection, mean positive b-wave amplitudes between Kv8.2KO mice treated with aav8.rk.hkcnv2 (Kv 8.2 KO-Tx), untreated Kv8.2KO mice (Kv 8.2 KO-UTx) and untreated wild type were compared. Data at two different stimulus intensities (10 and 25cd.s/m ²) are shown. Data are shown as mean ± SD and analyzed for statistical significance by two-way ANOVA with Turkey correction. * P <0.024; * P <0.003, < P <0.0003 and P <0.0001. As shown in this fig. 8, one subretinal injection of SEQ ID NO:2 and further injection of SEQ ID NO:3 was sufficient to significantly reduce supernormal b waves compared to untreated eyes. The b wave measures the time between the flash and the peak of the retina's response to a light stimulus or electrical activity. This data supports the restoration of photoreceptor function by SEQ ID NO:2 and further SEQ ID NO:3, as the retinal response time of treated Kv8.2KO animals was reduced over time compared to Kc8.2 untreated animals.

Retinal gene expression after treatment

Fig. 9 shows the relative gene expression of cones (a, cone arrestin) and rods (B, rhodopsin) markers in wild-type (WT), uninjected and treated retina P <0.05; * P <0.01. To further evaluate the efficacy of treatment with SEQ ID NO:2 and further with vector SEQ ID NO:3 for restoring photoreceptor function in a subject, such as a subject with CDSRR, for rod and cone cell health, gene expression levels of rhodopsin and cone cell inhibitor were evaluated. Rhodopsin is a marker of rod cell health. The inhibitor protein is a marker of cone cell health. Fig. 9 shows expression levels of rhodopsin and cone inhibitor in wild type, uninjected kv8.2 animals and injected kv8.2 animals. The data indicate that treatment with SEQ ID NO. 2 and further with SEQ ID NO. 3 results in gene expression of cone inhibitor protein. Cone arrestin showed a statistically significant increase in post-treatment gene expression levels compared to untreated retina. Although the rhodopsin level appears to be reduced in both treated and untreated retinas compared to WT, this is not statistically significant.

Turning to fig. 10, histological analysis and immunohistochemical labeling of kv8.2 subunits using antibodies to human KCVN peptide showed expression within the injected portion of the retina, while untreated areas did not show expression of kv8.2 protein. Specifically, FIG. 10 shows representative images of retinal expression of human Kv8.2 subunit in the retina of Kv8.2 KO mice injected with SEQ ID NO:2 delivered by SEQ ID NO:3 at 12 weeks post-treatment. (A) The wide view of the retinal slice is shown as the left treated area and the right untreated area. Scale bar = 200 μm. (i) High magnification inset of the treated area showing expression of human kv 8.2. (ii) No inset of untreated areas of kv8.2 expression are shown. Scale bar = 25 μm for (i) and (ii).

Functional ERG analysis of treated eyes compared to wild type

Figures 12 and 13 provide a comparison between treated and untreated mice using the disclosed methods, systems and nucleic acid sequences. To evaluate the efficacy of treatment with SEQ ID No. 2 delivered in the vector of SEQ ID No.3 to restore photoreceptor function in a subject (e.g., a subject with CDSRR), the a-wave amplitude and the positive b-wave ERG amplitude between treated (CDSRR animal represented by kv8.2 animal), untreated (CDSRR animal represented by kv8.2 animal) and wild-type eyes (wild-type animal) were compared. Quantification of scotopic (rod cell mediated) Electroretinogram (ERG) recordings of Wild Type (WT), kv8.2 KO untreated (uninjected) and Kv8.2 KO eyes treated subretinally with the 5e9 viral genome of the therapeutic product AAV8.RK. HKCNV2 (SEQ ID NO: 3). Records were obtained at 4 weeks post-treatment or for equivalent age-matched controls. WT, n=12; not injected, n=11; aav8.rk.hkcnv2, n=12. Two-way ANOVA with Tukey multiple comparison test, < p <0.05, < p <0.005, < p <0.0005 and < p <0.0001. The data indicate that treatment with SEQ ID NO:2 delivered in the vector of SEQ ID NO:3 restores photoreceptor function in a subject, such as a subject with CDSRR represented by a Kv8.2 KO animal.

FIG. 14 shows quantification of scotopic oscillation potential 1 (OP 1) at 25cd.s/m ² for Kv8.2KO eyes from Wild Type (WT), kv8.2KO untreated (uninjected) and subretinal treatment with the 3e9 viral genome of the treatment product AAV8. RK.hKCNV2. Records were obtained at 12 weeks post-treatment or for equivalent age-matched controls. WT, n=10; not injected, n=6; aav8.rk.hkcnv2, n=6. Two-way ANOVA with Tukey multiple comparison test, × p <0.0001. The data indicate that treatment with SEQ ID NO:2 delivered in the vector of SEQ ID NO:3 restores photoreceptor function in a subject, such as a subject with CDSRR represented by a Kv8.2KO animal.

FIG. 15 shows quantification of c-waves from Wild Type (WT), kv8.2KO untreated (uninjected) and Kv8.2KO eyes treated subretinally with the 5e9 viral genome of the treatment product AAV8.RK. HKCNV2. Records were obtained at 4 weeks post-treatment or for equivalent age-matched controls. WT, n=12; not injected, n=9; aav8.Rk. Hccrclv 2, n=15. Two-way ANOVA with Tukey multiple comparison test, <0.0001. The data indicate that treatment with SEQ ID NO:2 delivered in the vector of SEQ ID NO:3 restores photoreceptor function in a subject, such as a subject with CDSRR represented by a Kv8.2KO animal.

Apparent motor behavior response

Fig. 16 and 17 show improved photopic and scotopic visual acuity and scotopic contrast sensitivity in kv8.2ko mice treated 12 weeks after treatment. Treated animals were given subretinally 3e9 viral genome doses of aav8.rk.hkcnv2 (n=3-6) and compared to age-matched untreated (non-injected, n=3-5) and wild type (n=8) animals. Two-way ANOVA, posthoc multiple comparison of Sidak (Sidak's multiple comparison post-hoc), =0.0015, p <0.0001.WT was not significant with the treated aav8.Rk. Hkcnv2.

Gene and protein expression data

Fig. 18, 19 and 20 are histological data showing kv8.2 protein and KCNV gene expression in treated kv8.2 KO eyes at 12 weeks post-treatment. Fig. 18 is an overview of retinal sections from subretinal injected eyes with treated areas showing human kv8.2 subunit (green) expression and untreated areas without kv8.2 expression. Fig. 19 provides higher magnification images showing treated and untreated areas of kv8.2 expression (green), kv2.1 expression (red) and nuclei (blue). Scale bar = 50 μm. Figure 20 provides data from real-time quantitative PCR showing expression of the human KCNV gene in treated eyes normalized to wild type. N=3 eyes.

FIGS. 21 and 22 provide sequence alignments demonstrating a comparison of the human KCVN coding region (SEQ ID NO: 12) and the optimized KCVN2 (SEQ ID NO: 2) disclosed herein. The uplink contains the nucleic acid of human KCVN (SEQ ID NO: 12). Downstream the nucleic acid comprising SEQ ID NO. 2. SEQ ID NO. 2 has about 76% identity with human SEQ ID NO. 12.

It will be apparent that variations or modifications may be made to the precise details of the methods or compositions described without departing from the spirit of the described embodiments. We claim all such modifications and variations that fall within the scope and spirit of the following claims.

Sequence listing

<110> Altema treatment Co (ARTEMA THERAPEUTICS INC.)

<120> KCNV <2 > variants and uses thereof

<130> 209-301PCT

<150> US 63/191,106

<151> 2021-05-20

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 13728

<212> DNA

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(13728)

<223> Human KCVN2 (full nucleotide sequence)

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cctaagagcg aggttgaccg acatttttat tagcaataat ctctgccttc ttctgattac 480

ctagagattt aagaccacat aatcatcctc tacctcacag ggtcaaggga gtgggggagg 540

aaatgggcta agaggttcta aatccctcct aacacttgct tcttccaaat cagcaagatt 600

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ctctctaaga cagtgcaggc cacgtgatcc atcctcctag aggcagtgag caggtgaggg 780

acccctacca cagccaggag gaaaaagcta ggcgtccact ttccgcagcc atgctcaaac 840

agagtgagag gagacggtcc tggagctaca ggccctggaa cacgacggag aatgagggca 900

gccaacaccg caggagcatt tgctccctgg gtgcccgttc cggctcccag gccagcatcc 960

acggctggac agagggcaac tataactact acatcgagga agacgaagac ggcgaggagg 1020

aggaccagtg gaaggacgac ctggcagaag aggaccagca ggcaggggag gtcaccaccg 1080

ccaagcccga gggccccagc gaccctccgg ccctgctgtc cacgctgaat gtgaacgtgg 1140

gtggccacag ctaccagctg gactactgcg agctggccgg cttccccaag acgcgcctag 1200

gtcgcctggc cacctccacc agccgcagcc gccagctaag cctgtgcgac gactacgagg 1260

agcagacaga cgaatacttc ttcgaccgcg acccggccgt cttccagctg gtctacaatt 1320

tctacctgtc cggggtgctg ctggtgctcg acgggctgtg tccgcgccgc ttcctggagg 1380

agctgggcta ctggggcgtg cggctcaagt acacgccacg ctgctgccgc atctgcttcg 1440

aggagcggcg cgacgagctg agcgaacggc tcaagatcca gcacgagctg cgcgcgcagg 1500

cgcaggtcga ggaggcggag gaactcttcc gcgacatgcg cttctacggc ccgcagcggc 1560

gccgcctctg gaacctcatg gagaagccat tctcctcggt ggccgccaag gccatcgggg 1620

tggcctccag caccttcgtg ctcgtctccg tggtggcgct ggcgctcaac accgtggagg 1680

agatgcagca gcactcgggg cagggcgagg gcggcccaga cctgcggccc atcctggagc 1740

acgtggagat gctgtgcatg ggcttcttca cgctcgagta cctgctgcgc ctagcctcca 1800

cgcccgacct gaggcgcttc gcgcgcagcg ccctcaacct ggtggacctg gtggccatcc 1860

tgccgctcta ccttcagctg ctgctcgagt gcttcacggg cgagggccac caacgcggcc 1920

agacggtggg cagcgtgggt aaggtgggtc aggtgttgcg cgtcatgcgc ctcatgcgca 1980

tcttccgcat cctcaagctg gcgcgccact ccaccggact gcgtgccttc ggcttcacgc 2040

tgcgccagtg ctaccagcag gtgggctgcc tgctgctctt catcgccatg ggcatcttca 2100

ctttctctgc ggctgtctac tctgtggagc acgatgtgcc cagcaccaac ttcactacca 2160

tcccccactc ctggtggtgg gccgcggtga gtacctttgc cctgggcttt cccatcctct 2220

tccccagccc agtgagctgc tcctccctcc cctggttatc agccaccagg ctttggcttc 2280

tgatcctcgt cttccccccc acccccaatc gccgcataca gctaacaaaa cggcgatgga 2340

tgtcaaaagt ggtggaaaga gaactcagca gatcagtaag taagtgaatt tgacttagtc 2400

gtagaaatct ccaaatctag atttcgtctt caaaccttta aaagacaggt tttaaagaag 2460

atgcgtcatc attactgtta tttaccagtt attgagcatc cagtgtcctg acaaagctta 2520

tctcattgtg gcatcacagc ctttgagatg gttatgacca ccattttttt ttaaggggac 2580

aagcagaaat ggcttcagtt tttgaaagaa actgtagatt tatacagaga aggaggtgag 2640

acttggctga accatgttga ctctaactga aatcccacca cctctggttc cacttaaatc 2700

tgagtgtgga aagaagcatc tcaaactgaa acttgctctg acttcactaa agttctttca 2760

ggaccctgta ttattgcccc attttacaga tagtgaaatt gaagtacaga gagtttgaat 2820

gtggactcac agctagaggt ggcaaagcca gaactgaaac tcagtcctgt ccatctccaa 2880

actccatgcc tttcccacac ccaagcacag tctctttcaa gttttagttc ttttgcatgc 2940

attgctgaat ttgtacagtt agtgtaacag ttattttttg tcattgtttt ctttggcctt 3000

tgccttttta taacttgtgc tgtttactca aatatctgta ttttgagctg tggtagctga 3060

atccctggaa ataaatgtta atcaggtctt ctcagatcga tgaataagtc ggcatatatg 3120

aaggaaagaa ttgaatgtat ggtttcctta gttttctttt gaaaagtaga ttgtaatacc 3180

tttaaagaca ttaagcaaat ataactagtt ttcccatgtc agaaagtaga tattttcaac 3240

attgtgtatg tcataaccca tagtctggtg tgcacttctg agacaaacca acccaaggcc 3300

actgacagtg caatgcagca taatagaaga tttagaaaac ctgattctag tcccagacct 3360

gtcaataatt aaccatgacc ctgaataatt aactgtgaaa ccttaatccc tcacttccag 3420

tcctgctttc tcacctctaa aaggggaggc tggacatgta aaagtctgca gttcagtgag 3480

tcttccttcc cttgaaagca gggagctcat atgtatagag gatatgtgag ctttcttcag 3540

tggcaggatg atacgatgtt cttctgaatg agctaggcaa attctgcttc tctgatcata 3600

ttccaagtga tgaaaggtca tccctatagt agataaacca tgtgtacaga tgaggctcat 3660

ggttgatgat ttgtccccag caaatgacat ttactttggg gtgactaaag caggaaagca 3720

gaagagtaaa tccacatgct gatgatatga tacaacactt gagagtcaaa tttctattca 3780

tctaaacatc tttaaaggct agtgcatgaa ggcactggag ctagtacact ctctgatact 3840

acagtttgaa cccacaaaag ttttttcgac cttttgtata ttaacaaata ctttgataaa 3900

agttatgtta ctttcaaacg gatatggcta gagaaaaaat tttaattaaa ataaaacaca 3960

taaagaataa tgttactcag gttgtatttt taccagtaaa tttttttgaa ctgaataccc 4020

attgatctca cttaaatttc tgatctgttc aatgtacaat gaagtctaaa cctttcaaat 4080

tatctctttt tatgttaact ggcactatgt aactcaaaat caatgttttt cataaatttt 4140

acaaaaattg aatcaccagt ttttgccaat cataaatgtt accaattatt gctcttatct 4200

gtatgaattt ccttttgaag ccattatctc tatcagccac tattttcata ggcccaattt 4260

tgcacattcc aaccatccca tttacaaacc ttacccttta tcaatggtct ttgtccattt 4320

tattctgaaa attatttata ccagtaaaat tcgttacaaa ttagataaaa gactgatcat 4380

ctgtatttat ttctaaacta atctatgaaa acaatgggag caattgccct tgaatattct 4440

aagaccgctc tgttccccag gactgctatg ttattaatcc agttcttctc tgtacttcta 4500

actacactcc ataatacaga ctaaaggttc atattctcaa tagagcaaag agaaaagaac 4560

acattgaatt gatgtggaca aaatgtcttg tttccggtat gtagatgcaa agtgaaaaga 4620

aacaaaacat taggaccaat ttgtcatata acccacaaaa attcacctta ttcattcatt 4680

gaacagtaag tttttgagca cctccaatgt gcccagtagt attcttaaca ctagggatac 4740

agggtaggag aagaaaaaac aatcaagttt tttctcttct ggagcttaca ttctaatgga 4800

gaaagacaga taataaataa tttcaggtat tggcatagct tgtgtgggat aacaagattg 4860

agagtgattc aggcagtaga gtctgtcttt agtgggatag tcagagaagc ccttttggag 4920

aagatgacat tgagataaag tttgaaagat gaaaagagac tcatctaaat gcacctgact 4980

caacagatat gacaatctga gaagaggatt cctaaaagta actgatctag tggtttctaa 5040

gcattttatt aatcatgcat cttattttta aaaatttggg gcacataccc atgataaatg 5100

tgtatttatt tataaataaa ttagaagtta tttatttata aataaattag aagttattta 5160

taaataaatt agaagttatt tataaataaa ttagaagtta tttataaata aattagaagt 5220

tatttataaa taaattagaa gttatttata aataaattag aagttattta taaataaaat 5280

agaagttatt tatttataaa taaattagaa gttatttatt tataaataaa ttagaagtta 5340

tttatttaca aattagaagt tatttattta caaattagaa gttatttata aattagaagt 5400

tatttataaa taaattagaa gttatttata aataaattag aagttattta taaataaatt 5460

agaagttatt tataaataaa ttagaagtta tttataaata agttatttat aaatagaagt 5520

tatttataaa taagttattt ataaatagaa gttatttata aataagttat ttataaatag 5580

aagttattta taaataagtt atttataaat aaaagttatt tataaataaa ttagaagtta 5640

tttataaata aattagaagt tatttattta taaattagaa gttatttatt tataaataaa 5700

ttagaagtta tttatttata aataaattag aagttcttta ttgccataat aacgatacat 5760

caccaaccac cctgaaattc aatggcttaa aacatcaagc gtttatttag ctcatgaatt 5820

taaaagtcag cgtatactct gatttcagct agaaggttct tctctttcat gaggctcttg 5880

ttactcattc acatgcctga tggtcaactg gcatgaaggg agagactagg ccacatgtct 5940

gcattcctct ggcagactcc agcagccaca tgtcgatggc tgtggcaaag aacaagagag 6000

agaatgcaag ccccatcatg caaacacttc ataagtttct tttcatggca tttgctcagc 6060

ccattggcca gagtaagtca catggccaag ctgcaagtca aagggcaggg caggttagcc 6120

cacccaccat aggagggcac tgcaaagtta tatggcaaac agcttagaca cagaaagatg 6180

taaagaatta gttccaataa tgcaatcttt ctcctgcagt aatatattac tgctcaaata 6240

tattacacat atcagaaagt atacactcac aaattgaaac tttaaagatg agataaagaa 6300

aaatataatt ggcattttat ttcaccattc tggggctgta tcctttgtga aatatgcatc 6360

ccacattaca gagagtttct cttatccatt cttttatgta tttttaatac tttcagacaa 6420

caccaaatct gacttgcttt taataatctt tcttctctct atgccaagga tcctgcaaac 6480

tttctctgta attcaactca aggatttaag aaaaatacag tagttttcat tactgtgaat 6540

tctcctgcag ctgcaaaaca aatccctcct ggacacttat gtctacaaat gctcatgtat 6600

taggatgtag gatagacctg tctttttcat ctgtcaaatg gaggttattt gtgagcacta 6660

atgtaatagg aaacatatgt aaactgctag cctatagtaa gtctgcaata aatagaagct 6720

accataactg actagttgat agtctttgtc acatggaagc aataaccttt gcttctacat 6780

tcatttggga agaaaataaa acagaaatga ttctttttct actgaggaaa agaaagaacc 6840

tataaaacat acgagaaggt cagctgttcc ctcatgggag aacttctgag agtccagata 6900

aacaggccca cgcctaaagc agctccagcc atcttggggc tctctggatt gcctgggggt 6960

aggaaatgca cggcacagct aaacaagaac aatgggctct atgatgaagc tgtaatcatt 7020

ggcctcctgt gtgggtatgg tttttttctt ttactttttt ttttttttta gcctcaacat 7080

atgaccacat ttttttttct tatttttttt ttgccggggg ttggggggtg gagggcatgg 7140

gtatgttttc aagtagaaaa gagtaagtag actttgaatg aaccatttcc ttctgagttt 7200

tccagggcaa gttaaagctg tagaaaccaa acaggaaact acggcatgaa ctcactcctt 7260

caaaaactaa cttctcaaaa agaaaattta tctctagttg aggcaccatt ttgtcccaga 7320

gaccttgctc ccatctttct ttccctaagg tatgttttca ttaccttgcc aataggtggc 7380

acttacaaga tcactgaaat tattttattt ttgttcattg attgctttga ggaaacccat 7440

ttgaaaaata aattaggttg cagtcaggaa aatagatacc aaggtgaata tttcaaacag 7500

ggaatttaat acaaggaaat agatagttgt tagaagacta gaagaagaaa aaaaagaaga 7560

ctggaaaagc agaaagagaa aactgagata atccagagat tggtaattgc agggagtagc 7620

tattggccct agaattggga gaataaatgg gaaaaggtgt tgccaccaga agctaagagt 7680

ccacaggagg ggcctctgtg tagctgttgc aaggactcct aaattggcac catgaggctg 7740

atgccaggaa tgccaaaaaa tgcctgaacc atagctattg gctgcaattg gaggaatgaa 7800

ggcagaaacc agaggcagga ttagagaccc ttatgtcctc ataccatctt ccagtctctc 7860

ataggcagaa cataactaga agacagtggg caagagaatc tgggaaatgt agtttgcaga 7920

ctcccagttc tggaattata aaataaaata cagaagggtg tcaggcttgg gactaagaga 7980

caacatataa atatctggca cagaaagtaa tagagttcta cctccttcag aagtagcaca 8040

ggagctatag acggggtaaa ggctcttagg cattgttact actttttatc ttatggattc 8100

ccaattttgg agtggtctcc ttttatgtct ttaaagtatg ttcctcattt tagccaatat 8160

gatgtgagtg gaacatgaca gcatcattga gggcacggag aaatgcaggg aagctgaggg 8220

tgtggacagc atcattattg cgcacctttc tgtggataga ttaatagatc tcaaacttac 8280

atttgtggct ttgctttagc tgtgcacacc taccgcttat tgtaccactt acaaacatgt 8340

cagaactgta atatacagat gagggttgtt attatcctga aggtagtcac ccatgacctc 8400

attctaacag tgattccact aatcaagaca ttctttgaat tccactgagg aattaccttc 8460

agagtttgtg acacatccac ttgtttctag gttgttaaat ctttgtcttt ttaaggctac 8520

attcagtatt ttaaaatagt taagacactt tagtttaaga tcgacactta gagtaaacca 8580

tcaaggtgga tatgaccgct tgctgctact gaatctactg tgcagactat aattcaaaag 8640

aggaattcca aatatacttg ttgcaataat agcatgagta agtacagcct ttgaagctgg 8700

atcctttgaa ggacaacacg tattgaggct tataataatt ttatccaaaa aagatccatc 8760

ttcaccacct ctaattcctt tggaaaacag aaagagtgtt attttgaaaa ataagtttta 8820

ttccctagga tcataaattc ctgaatgcat ggactactgg gtgccaagat tgattccatt 8880

ccttgttcat tatgagggaa acaaacttgt cttgttaaat gagaaatcca agtattctca 8940

agttttggag aaaaatttga tgcttatcct cataatatat ggcaggccag gatttgttgt 9000

ttagtgagta aactgccagg ggaaggtcac attctggctt tgggacccaa ggctaaggaa 9060

agtatgtacg tttacagcag aatggttaag cttccagttt gcagagttgc tgagctagcc 9120

agttttcttc agtatattaa gatggattgc ggaaattttg gtcacatttc caaacccaac 9180

tcctgctaat gtgacaactc attctgtgag gtcacttggg tcagtctggg gccttcagct 9240

tttaattccc atctagggag aaatggcttg gcctatggaa tgtggaaaaa aagttcccca 9300

aagccaagga atcctgagtt cctcctggag ccaaggaaca agaaacagat cacttgttga 9360

gtcaaattat tcacgaaata gacaacatat aaattaactg tacattttca acaaattatt 9420

ggggaccagt cacacatagc ctcctagcag aagagatctg ttctctaaaa atagagattg 9480

aagatataca aaaatatttg gatcatatta tacacatcag atatttattc ttctttaagc 9540

caattctctt tgcttttgac ttttcactca aagttcagcc aaaaaaaaaa gaagtttggc 9600

ttaagggtga agcaaagcca gtgtgcagaa tgatcaaaag ttgaaaataa gcagtttatc 9660

ttaattattt tcttcctcag tgtcttccta ctctctccca tccccgccat cctgaaccct 9720

taagtgactt gtttcctcca aggcctctgc attggacttc cgtggatgtc tcatgttcta 9780

aaccaggggt ccccaatccc gggctgtgga gtggtacttg tctgtggcct gttaggaacc 9840

cggccacaca gcaggagatg agtggagggc catcaatcat taccgcctaa gttctgcctc 9900

ctgtcagatc aaccacagcg ttagattctc ataggagtgg gaaccctatt gttaactgcg 9960

catgggaggg atctaggttg cttgctcctt atgagaatct aactaatacc tgacaacctg 10020

agatggagca gtttcatccc gaaaccatcc cctcccccaa ccccgtccat gggaaaaatg 10080

gtcgtccaca aaaccggtcc ctggtgccaa agaggttggg gaccgctgtt ctaaactacc 10140

ccttcctgtc actcactcct aaatccaaac tccctttagg cctcctccag ggaaggttga 10200

ctaaactttc atccagaggt gtgagtcatg gtgttagtga caaatgaaag ctattgttca 10260

gctcacacaa ctggagagag atcagcctgg ggttctttga cttttggtgt ggtcaatgac 10320

aagaaacaca cacccggaca caggaagggg aacatcacac accagggcct gttgtggggt 10380

gggggagggg ggaggggtag catttggaga tatacctaac gttaaatgac aagttactgg 10440

gtgcagcaca ccaacatggc acatgtatac atatgtaact aacctgcacg ttgtgcacat 10500

gtaccctaga acttaaataa aaaaaagaca cacccactca tgcaaccaag aggactttga 10560

gattaaccca caatctcact ttatcactgc ctcacatcac cccttggtcc agaagttggg 10620

agaaggatat tctctactgg agccagaggg cagagtcagg gaggagcttg tttccacagc 10680

ctgagggtct gataagcata acgtatatag agttgctggt gtagaatgca ctcaataaat 10740

gttggtcttt gcctcttctc agtggaggcc agcttcagca tgggggaccc tgggattgca 10800

tacatgatat atatataata atattattgc ttacacaccc tgtgccaggc actccttcaa 10860

ttcctataac aactctgtga ggtagatacc acacatttca aaagtagaaa ctgaggccaa 10920

gagaatttag gtgatctgcc caacttattc agctgggaag tggattcaga cccacaagtc 10980

tactgtagtc cgtttccttc agcttatcta caccacatta cctctcttgg atgcctgctt 11040

gtattttgac tctgcagaaa aaggctgcat gacctctcac aagagacagg gcagtgagtg 11100

acaggaggtg catcatcagc ctcaaaaagg aggcataccc ttcctttttt cgtgggctgg 11160

atggagctgt ggttctcatc agtgccctca gttttgtgca gctggctcac ttctccattt 11220

cattctaatc agtcaactgc actattccag gttcaaacta acccttacat catcaaaaca 11280

aaaatattta cactgctaag ctaacggcca cctcagcacg gatcaacaag atgaccatat 11340

gctttactgc cgtctgctgg aaaattatgg caacaactac ccaaatacac aaagcaaaga 11400

gtgcccccct ggagtagtgc aacgcaaaaa tgtttacaac tgtgaccaac agctctgctt 11460

aaaaggcttc tagtaattta gccaatactc tggggatcag agggagtaca tgaggcataa 11520

aaaacagtcc ccaaggaatt ttcacagggt tttctctggt aactgataac tagtcccatg 11580

gtatctgcat tttaaaacag aagcttgtta acctaaataa gtctccaatt agtgggattt 11640

aaatcagtat gacaagagta atgggaagta tttcatgcag gggtgaacat atttttggtg 11700

agtgatgtct tacaaaagtc cctttacaaa ataatgagag tgtttctcca gacatctgca 11760

attaaagcac cttcacataa agtctctcat ttgaactacg taacaacttt gaaaagtgtt 11820

ataactgtct tcattttaaa gatgagagga agacaatgga ggagtttcat gccttttgta 11880

tgcctggtac tgtgttcagt gctttatatt cattttctta tttagccttc acaagaatcc 11940

taagagttag atggttttct cctgttttaa ataaaaaaaa aaaaaaagaa aaaaagaaaa 12000

agagagagag agagtctgaa ggttttgccc caggtcactc aactggaaag gttcagagtc 12060

aggacttgaa cccaggtctg actgttctaa gcccaagatt tttccaatac atacagtgta 12120

caggcaaacc cagggacctg ctttcctgaa tctggtgcca gctgagttag ggaggcaaag 12180

atcatttact gagcacgttc tacatcaggt acttaacata ctattttaaa tgctctttac 12240

agcaaccatt tcaagtaggt attacctcct cctcccatat cttacattca aacatgcatg 12300

agtcgtagtc aggatttcag ccaaagtctt tcagctccat ccatagcttc tgttcttttc 12360

atgacacagg tcctagaggg agtcttcctg gtacctccta aagcaggctc cgtgggaagc 12420

cattacactt cccatgtgta cccacaggga ggacgcttcc ctgcttgctc ctctcccttt 12480

cttctcctcc ccgatcttag tgctaacaat tccatcctgc tttccttcct ctacaggtga 12540

gcatctccac cgtgggctac ggagacatgt acccagagac ccacctgggc aggttttttg 12600

ccttcctctg cattgctttt gggatcattc tcaacgggat gcccatttcc atcctctaca 12660

acaagttttc tgattactac agcaagctga aggcttatga gtataccacc atacgcaggg 12720

agaggggaga ggtgaacttc atgcagagag ccagaaagaa gatagctgag tgtttgcttg 12780

gaagcaaccc acagctcacc ccaagacaag agaattagta ttttatagga catgtggctg 12840

gtagattcca tgaacttcaa ggcttcattg ctcttttttt aatcattatg attggcagca 12900

aaaggaaatg tgaagcagac atacacaaag gccatttcgt tcacaaagta ctgcctctag 12960

aaatactcat tttggcccaa actcagaatg tctcatagtt gctctgtgtt gtgtgaaaca 13020

tctgaccttc tcaatgacgt tgatattgaa aacctgaggg gagcaacagc ttagattttt 13080

cttgtagctt ctcgtggcat ctagctcaat aaatattttt ggacttgagt tgacttgaga 13140

aaattttttt tactttaaat ttttctaaaa ttcttaactt tccagaggga gggagggtta 13200

cagcagaaat tatacaagct ttggagttag accaacctta gtcagaatcc cagagctacc 13260

agctgtatgc ttttaggcaa gcgactctaa ccctttaagc ctcagtttct tcaactgtga 13320

aatgtaggca atacttaccc tgccaggctg agcaatatag tgagaccctg cctctacaaa 13380

aacttattaa gaattatctg ggtgtgctgg cccacacctg tagtcccagc tacttggaag 13440

actgaggtga gatcacttga gcccaggagt ttgaggttag agtaagctat aaccacatta 13500

ctgcactcca atctagatgg cagagtaaga ccctgcctca aataaataaa taaataaata 13560

aataaataaa accacctgcc ttgtaaaatg agtctgggga taacagatat gtgtaaacgc 13620

tcaataaatg ataggtatta aaattgttta agtggatgtt atctagtgaa atctctagac 13680

cagtggttct caaaggcaaa ttcattcctc agaggccagc taatgcct 13728

<210> 2

<211> 1638

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis: codon optimized hKCVN2 (nucleotide sequence)

<400> 2

atgctgaagc agagtgagag gaggaggtca tggagttatc gaccttggaa cacgactgaa 60

aacgaaggca gccagcatcg cagatccatt tgctccctgg gggcgcgcag tggctcacaa 120

gcgtccatcc acggctggac tgaaggaaat tataactact atatagagga ggacgaagac 180

ggagaggagg aagaccaatg gaaagatgat ctggcggagg aagatcagca agccggtgaa 240

gtgaccactg ctaaacccga aggaccatct gacccacctg cactcttgag cacattgaat 300

gtaaatgttg ggggtcacag ctaccaattg gattactgcg agcttgccgg gtttcccaag 360

actcggctcg gaaggctcgc aacatccaca agcaggtccc ggcaattgtc actgtgcgat 420

gactatgaag aacaaacaga cgagtatttc tttgacaggg acccggctgt cttccagttg 480

gtctataact tctatctgtc aggtgttctc ctcgttctcg atggcctgtg tcctcggcga 540

ttcttggaag aactcgggta ctggggggtg aggttgaaat atacccctcg gtgctgccgc 600

atttgttttg aggaaaggcg agatgagctt tcagagcggt tgaagataca acacgaactt 660

agagcgcagg ctcaggtaga agaagctgaa gaattgtttc gagacatgag attttatggc 720

ccacagcgcc gccggctgtg gaacctcatg gaaaagcctt tctcaagtgt cgccgccaag 780

gctattggcg ttgccagcag cactttcgta cttgtgagcg tagtggcact ggcattgaat 840

actgtagagg agatgcagca gcacagcgga cagggtgaag gggggcctga ccttcggcct 900

atcctcgaac atgtcgaaat gctctgcatg gggtttttca ccttggagta ccttcttcga 960

cttgcatcta cgccagactt gcggagattt gctaggagcg ctcttaacct ggttgacctc 1020

gtcgcgatcc tgccgttgta cctccagctg cttctcgagt gttttacagg tgagggtcac 1080

caacgcggcc agactgtcgg gagcgtcgga aaggttggtc aggttctgcg cgtcatgaga 1140

ttgatgagga tatttagaat cctcaaattg gctagacata gtactgggtt gcgcgcattc 1200

ggtttcaccc ttcgacagtg ctatcagcaa gttgggtgct tgctcttgtt catcgctatg 1260

ggaatcttca ctttttccgc cgccgtatat tccgtagaac atgacgttcc ctccaccaat 1320

tttacaacaa tcccgcatag ctggtggtgg gctgctgtct ccatctctac ggtcggctac 1380

ggcgacatgt accccgaaac gcacctcggt aggttcttcg catttctgtg catcgcgttt 1440

ggaatcattc ttaatggtat gcctatttca atactttaca ataaattctc cgattactac 1500

agtaaattga aagcatacga gtatactacg attcggcgcg agaggggcga agtaaatttc 1560

atgcagcgag caagaaaaaa aattgccgag tgtctgctgg ggagtaatcc acagctcaca 1620

ccacgccaag aaaactag 1638

<210> 3

<211> 5971

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis: the complete DNA sequence of the construct,

PAAV_RK_ KOZAK _ hKCNV2_WPRE-SV40 full sequence

<400> 3

gatatcctta agggatccag atctgaattc gggccccaga agcctggtgg ttgtttgtcc 60

ttctcagggg aaaagtgagg cggccccttg gaggaagggg ccgggcagaa tgatctaatc 120

ggattccaag cagctcaggg gattgtcttt ttctagcacc ttcttgccac tcctaagcgt 180

cctccgtgac cccggctggg atttagcctg gtgctgtgtc agccccggtc tcccaggggc 240

ttcccagtgg tccccaggaa ccctcgacag ggcccggtct ctctcgtcca gcaagggcag 300

ggacgggcca caggccaagg gcggtacgcc gccaccatgc tgaagcagag tgagaggagg 360

aggtcatgga gttatcgacc ttggaacacg actgaaaacg aaggcagcca gcatcgcaga 420

tccatttgct ccctgggggc gcgcagtggc tcacaagcgt ccatccacgg ctggactgaa 480

ggaaattata actactatat agaggaggac gaagacggag aggaggaaga ccaatggaaa 540

gatgatctgg cggaggaaga tcagcaagcc ggtgaagtga ccactgctaa acccgaagga 600

ccatctgacc cacctgcact cttgagcaca ttgaatgtaa atgttggggg tcacagctac 660

caattggatt actgcgagct tgccgggttt cccaagactc ggctcggaag gctcgcaaca 720

tccacaagca ggtcccggca attgtcactg tgcgatgact atgaagaaca aacagacgag 780

tatttctttg acagggaccc ggctgtcttc cagttggtct ataacttcta tctgtcaggt 840

gttctcctcg ttctcgatgg cctgtgtcct cggcgattct tggaagaact cgggtactgg 900

ggggtgaggt tgaaatatac ccctcggtgc tgccgcattt gttttgagga aaggcgagat 960

gagctttcag agcggttgaa gatacaacac gaacttagag cgcaggctca ggtagaagaa 1020

gctgaagaat tgtttcgaga catgagattt tatggcccac agcgccgccg gctgtggaac 1080

ctcatggaaa agcctttctc aagtgtcgcc gccaaggcta ttggcgttgc cagcagcact 1140

ttcgtacttg tgagcgtagt ggcactggca ttgaatactg tagaggagat gcagcagcac 1200

agcggacagg gtgaaggggg gcctgacctt cggcctatcc tcgaacatgt cgaaatgctc 1260

tgcatggggt ttttcacctt ggagtacctt cttcgacttg catctacgcc agacttgcgg 1320

agatttgcta ggagcgctct taacctggtt gacctcgtcg cgatcctgcc gttgtacctc 1380

cagctgcttc tcgagtgttt tacaggtgag ggtcaccaac gcggccagac tgtcgggagc 1440

gtcggaaagg ttggtcaggt tctgcgcgtc atgagattga tgaggatatt tagaatcctc 1500

aaattggcta gacatagtac tgggttgcgc gcattcggtt tcacccttcg acagtgctat 1560

cagcaagttg ggtgcttgct cttgttcatc gctatgggaa tcttcacttt ttccgccgcc 1620

gtatattccg tagaacatga cgttccctcc accaatttta caacaatccc gcatagctgg 1680

tggtgggctg ctgtctccat ctctacggtc ggctacggcg acatgtaccc cgaaacgcac 1740

ctcggtaggt tcttcgcatt tctgtgcatc gcgtttggaa tcattcttaa tggtatgcct 1800

atttcaatac tttacaataa attctccgat tactacagta aattgaaagc atacgagtat 1860

actacgattc ggcgcgagag gggcgaagta aatttcatgc agcgagcaag aaaaaaaatt 1920

gccgagtgtc tgctggggag taatccacag ctcacaccac gccaagaaaa ctagaagctt 1980

atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat 2040

gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct 2100

tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag 2160

gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc 2220

cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc 2280

ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct 2340

cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg 2400

ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg 2460

gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg 2520

cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga 2580

taccgtcgac ctcgacccgg gcggccgctt cgagcagaca tgataagata cattgatgag 2640

tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat 2700

gctattgctt tatttgtaac cattataagc tgcaataaac aagttgctag cctgcagact 2760

agttctagag atatcatctt cctagagcat ggctacgtag ataagtagca tggcgggtta 2820

atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 2880

tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc 2940

tcagtgagcg agcgagcgcg cagccttaat taacctaatt cactggccgt cgttttacaa 3000

cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 3060

ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 3120

agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg 3180

gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc 3240

ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc 3300

cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt 3360

gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag 3420

tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg 3480

gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag 3540

ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttat aatttcaggt 3600

ggcatctttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 3660

aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 3720

aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 3780

cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 3840

ggtgcacgag tgggttacat cgaactggat ctcaatagtg gtaagatcct tgagagtttt 3900

cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 3960

ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 4020

gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 4080

gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 4140

acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 4200

cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 4260

acgatgcctg tagtaatggt aacaacgttg cgcaaactat taactggcga actacttact 4320

ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 4380

ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 4440

gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 4500

atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 4560

ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 4620

attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 4680

ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 4740

aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 4800

aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 4860

ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 4920

tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 4980

ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 5040

cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 5100

agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 5160

gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 5220

ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 5280

tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 5340

tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg cggttttgct 5400

cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 5460

tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 5520

gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc 5580

agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg 5640

agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg 5700

tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc 5760

agatttaatt aaggctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc 5820

gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg 5880

ccaactccat cactaggggt tccttgtagt taatgattaa cccgccatgc tacttatcta 5940

cgtagccatg ctctaggaag atcggaattc g 5971

<210> 4

<211> 130

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis: 5 ITR nucleotide sequence

<400> 4

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct 130

<210> 5

<211> 130

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis: 3 ITR nucleotide sequence

<400> 5

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120

gagcgcgcag 130

<210> 6

<211> 297

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis: RK promoter

<400> 6

gggccccaga agcctggtgg ttgtttgtcc ttctcagggg aaaagtgagg cggccccttg 60

gaggaagggg ccgggcagaa tgatctaatc ggattccaag cagctcaggg gattgtcttt 120

ttctagcacc ttcttgccac tcctaagcgt cctccgtgac cccggctggg atttagcctg 180

gtgctgtgtc agccccggtc tcccaggggc ttcccagtgg tccccaggaa ccctcgacag 240

ggcccggtct ctctcgtcca gcaagggcag ggacgggcca caggccaagg gcggtac 297

<210> 7

<211> 583

<212> DNA

<213> Human cytomegalovirus

<220>

<221> misc_feature

<222> (1)..(583)

<223> CMV promoter nucleotide sequence

<400> 7

tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360

catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420

atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480

ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540

acggtgggag gtctatataa gcagagctgg tttagtgaac cgt 583

<210> 8

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis: KOZAK nucleotide sequence

<400> 8

gccgccacc 9

<210> 9

<211> 542

<212> DNA

<213> Woodchuck hepatitis Virus

<220>

<221> misc_feature

<222> (1)..(542)

<223> Woodcuck hepatitis Virus (WHP) post transcriptional regulatory element (WPRE)

<400> 9

aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60

ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120

atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180

tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240

ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300

attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360

ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420

gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480

aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540

cg 542

<210> 10

<211> 215

<212> DNA

<213> Cattle

<220>

<221> misc_feature

<222> (1)..(215)

<223> Bovine growth hormone polyadenylation (BGH) poly (A)

<400> 10

gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 60

ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 120

cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 180

gaggattggg aagacaatag caggcatgct gggga 215

<210> 11

<211> 545

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(545)

<223> KCVN amino acid sequence

<400> 11

Met Leu Lys Gln Ser Glu Arg Arg Arg Ser Trp Ser Tyr Arg Pro Trp

1 5 10 15

Asn Thr Thr Glu Asn Glu Gly Ser Gln His Arg Arg Ser Ile Cys Ser

20 25 30

Leu Gly Ala Arg Ser Gly Ser Gln Ala Ser Ile His Gly Trp Thr Glu

35 40 45

Gly Asn Tyr Asn Tyr Tyr Ile Glu Glu Asp Glu Asp Gly Glu Glu Glu

50 55 60

Asp Gln Trp Lys Asp Asp Leu Ala Glu Glu Asp Gln Gln Ala Gly Glu

65 70 75 80

Val Thr Thr Ala Lys Pro Glu Gly Pro Ser Asp Pro Pro Ala Leu Leu

85 90 95

Ser Thr Leu Asn Val Asn Val Gly Gly His Ser Tyr Gln Leu Asp Tyr

100 105 110

Cys Glu Leu Ala Gly Phe Pro Lys Thr Arg Leu Gly Arg Leu Ala Thr

115 120 125

Ser Thr Ser Arg Ser Arg Gln Leu Ser Leu Cys Asp Asp Tyr Glu Glu

130 135 140

Gln Thr Asp Glu Tyr Phe Phe Asp Arg Asp Pro Ala Val Phe Gln Leu

145 150 155 160

Val Tyr Asn Phe Tyr Leu Ser Gly Val Leu Leu Val Leu Asp Gly Leu

165 170 175

Cys Pro Arg Arg Phe Leu Glu Glu Leu Gly Tyr Trp Gly Val Arg Leu

180 185 190

Lys Tyr Thr Pro Arg Cys Cys Arg Ile Cys Phe Glu Glu Arg Arg Asp

195 200 205

Glu Leu Ser Glu Arg Leu Lys Ile Gln His Glu Leu Arg Ala Gln Ala

210 215 220

Gln Val Glu Glu Ala Glu Glu Leu Phe Arg Asp Met Arg Phe Tyr Gly

225 230 235 240

Pro Gln Arg Arg Arg Leu Trp Asn Leu Met Glu Lys Pro Phe Ser Ser

245 250 255

Val Ala Ala Lys Ala Ile Gly Val Ala Ser Ser Thr Phe Val Leu Val

260 265 270

Ser Val Val Ala Leu Ala Leu Asn Thr Val Glu Glu Met Gln Gln His

275 280 285

Ser Gly Gln Gly Glu Gly Gly Pro Asp Leu Arg Pro Ile Leu Glu His

290 295 300

Val Glu Met Leu Cys Met Gly Phe Phe Thr Leu Glu Tyr Leu Leu Arg

305 310 315 320

Leu Ala Ser Thr Pro Asp Leu Arg Arg Phe Ala Arg Ser Ala Leu Asn

325 330 335

Leu Val Asp Leu Val Ala Ile Leu Pro Leu Tyr Leu Gln Leu Leu Leu

340 345 350

Glu Cys Phe Thr Gly Glu Gly His Gln Arg Gly Gln Thr Val Gly Ser

355 360 365

Val Gly Lys Val Gly Gln Val Leu Arg Val Met Arg Leu Met Arg Ile

370 375 380

Phe Arg Ile Leu Lys Leu Ala Arg His Ser Thr Gly Leu Arg Ala Phe

385 390 395 400

Gly Phe Thr Leu Arg Gln Cys Tyr Gln Gln Val Gly Cys Leu Leu Leu

405 410 415

Phe Ile Ala Met Gly Ile Phe Thr Phe Ser Ala Ala Val Tyr Ser Val

420 425 430

Glu His Asp Val Pro Ser Thr Asn Phe Thr Thr Ile Pro His Ser Trp

435 440 445

Trp Trp Ala Ala Val Ser Ile Ser Thr Val Gly Tyr Gly Asp Met Tyr

450 455 460

Pro Glu Thr His Leu Gly Arg Phe Phe Ala Phe Leu Cys Ile Ala Phe

465 470 475 480

Gly Ile Ile Leu Asn Gly Met Pro Ile Ser Ile Leu Tyr Asn Lys Phe

485 490 495

Ser Asp Tyr Tyr Ser Lys Leu Lys Ala Tyr Glu Tyr Thr Thr Ile Arg

500 505 510

Arg Glu Arg Gly Glu Val Asn Phe Met Gln Arg Ala Arg Lys Lys Ile

515 520 525

Ala Glu Cys Leu Leu Gly Ser Asn Pro Gln Leu Thr Pro Arg Gln Glu

530 535 540

Asn

545

<210> 12

<211> 1638

<212> DNA

<213> Chile person

<220>

<221> misc_feature

<222> (1)..(1638)

<223> Human KCVN2 (coding region nucleotide sequence)

<400> 12

atgctcaaac agagtgagag gagacggtcc tggagctaca ggccctggaa cacgacggag 60

aatgagggca gccaacaccg caggagcatt tgctccctgg gtgcccgttc cggctcccag 120

gccagcatcc acggctggac agagggcaac tataactact acatcgagga agacgaagac 180

ggcgaggagg aggaccagtg gaaggacgac ctggcagaag aggaccagca ggcaggggag 240

gtcaccaccg ccaagcccga gggccccagc gaccctccgg ccctgctgtc cacgctgaat 300

gtgaacgtgg gtggccacag ctaccagctg gactactgcg agctggccgg cttccccaag 360

acgcgcctag gtcgcctggc cacctccacc agccgcagcc gccagctaag cctgtgcgac 420

gactacgagg agcagacaga cgaatacttc ttcgaccgcg acccggccgt cttccagctg 480

gtctacaatt tctacctgtc cggggtgctg ctggtgctcg acgggctgtg tccgcgccgc 540

ttcctggagg agctgggcta ctggggcgtg cggctcaagt acacgccacg ctgctgccgc 600

atctgcttcg aggagcggcg cgacgagctg agcgaacggc tcaagatcca gcacgagctg 660

cgcgcgcagg cgcaggtcga ggaggcggag gaactcttcc gcgacatgcg cttctacggc 720

ccgcagcggc gccgcctctg gaacctcatg gagaagccat tctcctcggt ggccgccaag 780

gccatcgggg tggcctccag caccttcgtg ctcgtctccg tggtggcgct ggcgctcaac 840

accgtggagg agatgcagca gcactcgggg cagggcgagg gcggcccaga cctgcggccc 900

atcctggagc acgtggagat gctgtgcatg ggcttcttca cgctcgagta cctgctgcgc 960

ctagcctcca cgcccgacct gaggcgcttc gcgcgcagcg ccctcaacct ggtggacctg 1020

gtggccatcc tgccgctcta ccttcagctg ctgctcgagt gcttcacggg cgagggccac 1080

caacgcggcc agacggtggg cagcgtgggt aaggtgggtc aggtgttgcg cgtcatgcgc 1140

ctcatgcgca tcttccgcat cctcaagctg gcgcgccact ccaccggact gcgtgccttc 1200

ggcttcacgc tgcgccagtg ctaccagcag gtgggctgcc tgctgctctt catcgccatg 1260

ggcatcttca ctttctctgc ggctgtctac tctgtggagc acgatgtgcc cagcaccaac 1320

ttcactacca tcccccactc ctggtggtgg gccgcggtga gcatctccac cgtgggctac 1380

ggagacatgt acccagagac ccacctgggc aggttttttg ccttcctctg cattgctttt 1440

gggatcattc tcaacgggat gcccatttcc atcctctaca acaagttttc tgattactac 1500

agcaagctga aggcttatga gtataccacc atacgcaggg agaggggaga ggtgaacttc 1560

atgcagagag ccagaaagaa gatagctgag tgtttgcttg gaagcaaccc acagctcacc 1620

ccaagacaag agaattag 1638

Claims (14)

1. A modified KCVN nucleotide sequence shown in SEQ ID No. 2 or a sequence having at least 85% sequence identity to SEQ ID No. 2 or a sequence having at least 90% sequence identity to SEQ ID No. 2, said sequence encoding a peptide of SEQ ID No. 11 and said sequence being capable of restoring photoreceptor activity.

2. A vector comprising the modified KCVN nucleotide sequence of claim 1.

3. The vector of claim 2, wherein the vector is a viral vector.

4. The vector of claim 3, wherein the viral vector is an AAV vector.

5. The vector of claim 2, wherein the vector is a gamma-retroviral vector, a lentiviral vector, or an adenoviral vector.

6. The vector according to claim 4, which has the nucleotide sequence of SEQ ID NO. 3.

7. The vector of claim 4, further comprising an RK promoter operably linked to SEQ ID NO. 2.

8. The carrier of claim 6, further comprising at least one of: KOZAK consensus sequences, woodchuck hepatitis virus (WHP) post-transcriptional regulatory elements (WPRE) and bovine growth hormone polyadenylation (BGH-poly (A)) signals.

9. The carrier of claim 7, wherein the elements are arranged in the following order from 5 'to 3': the RK promoter is then followed by KOZAK sequences, then SEQ ID NO:2, then WPRE, then BGH poly (A) signal.

10. A KCVN nucleotide sequence, said KCVN nucleotide sequence hybridizes under stringent conditions to SEQ ID No.2 and encodes a protein of SEQ ID No. 11 having photoreceptor activity.

11. The KCVN nucleotide sequence according to claim 9, the KCVN nucleotide sequence is operably linked to an RK promoter, KOZAK sequence, WPRE and BGH poly (a) signals.

12. A pharmaceutical composition comprising the vector of claim 2 in a pharmaceutically acceptable vehicle.

13. The pharmaceutical composition of claim 11, formulated for local, systemic, or topical administration.

14. The pharmaceutical composition of claim 12, formulated for oral, nasal, pulmonary, buccal, transdermal, subcutaneous, intraduodenal, enteral, parenteral, intravenous, or intramuscular administration.